CN1832773A - Methods for repairing and regenerating human dura mater - Google Patents

Abstract

A method of using a substantially non-porous equine collagen foil to repair and regenerate dura mater tissue of mammals when the dura mater tissue is damaged as a result of injury, tumors, surgery, and the like. The non-porous equine collagen foil comprises collagen fibrils which provides a replacement dura mater composition that is elastic, liquid-tight, and which has a high tensile strength. The non-porous equine collagen foil is furthermore resorbable and provides a biomatrix, wherein a neodura is rapidly formed which becomes indistinguishable from the autologous dura mater in a matter of weeks. The process for making the equine collagen foil reduces the likelihood of disease transmission.

Description

Be used to repair and the compositions of the people's dura mater of regenerating

The part of this patent file disclosure comprises material protected by copyright.The copyright holder does not oppose that anyone duplicates described patent document or patent disclosure content, described patent document or patent disclosure content is as in patent document that appears at patent and trademark office (Patent and Trademark Office) or the record, but keeps every other copyright in addition.

Invention field

The collagen thin slice compositions that the present invention relates to atresia substantially is as graft materials, to repair and/or the purposes of the mammiferous dura mater tissue of regenerating.More particularly, the collagen thin slice compositions that the present invention relates to inhuman source is the dura mater material as an alternative, and as the purposes of the regenerated bio-matrix of dura mater.

Background of invention

Dura mater is the structure that plays an important role in central nervous system's anatomy, can form bag by whole central nervous system's film system, and the protection central nervous system avoids external action.

Dura mater may be because multiple former thereby need to repair, and described reason comprises wound, inflammation or neoplastic process, operation process or congenital anomaly.Particularly after operation and after having wound during fistula to sealing the needs of dura mater defective, the needs to ideal dura mater succedaneum have been proposed.Above-mentioned defective may cause postoperative complication, the cerebral fit that specifically, cerebrospinal fluid oozes out, infection and result cause.The grafting of dura process of some forms is almost all needing in 30% the craniotomy.Because the preliminary closure of dura mater defective is normally failed, so be used to avoid the effectiveness of the dura mater succedaneum of above-mentioned complication to have important practice value.

So that take out after the malignant tumor in brain or the spinal column, need realize permanent liquid-tight closure to dura mater, in head injury or surgical intervention so that avoid cerebrospinal fluid to leak.The common use of neurosurgeon can be resorbent or can not resorbent dura mater succedaneum, and connects dura mater at skull or spinal column with stitching thread and/or fibrin adhesive ﹠ usually.Can resorbent examples of material comprise people's corpse dura mater, people's fascia lata, bovine pericardium, xenogenesis collagen sponge and from the implant of textile material, it is by forming by resorbent polyester (polyglactin and/or poly-p-di-oxanon).Example that can not resorbent dura mater succedaneum comprises the material of being made by polytetrafluoroethylene (PTFE) or polyester urethane.

Nearly all grafting of dura thing of studying up to now is all relevant with complication, and some are relevant bigger.The main complication of reporting is chronic inflammatory disease and rejection, and the formation of cortex meninges (corticomeningeal) adhesion, and it has caused the development of epileptogaenic focus.Also observed hematoma and cerebrospinal fluid fistula, the latter provides the various inlet points that cause the biology of disease conversely again.

Already multiple material and method were assessed in the past few decades,, comprised people's corpse dura mater of various metals, implant, synthetic material, autograft thing and preservation so that seek ideal grafting of dura thing.Major part in the said goods is inappropriate, and this is that some complication wherein is serious because of associated postoperative complication.The example of complication comprises that the development of chronic inflammatory disease and rejection, cortex cerebral meningeal adhesions, hemorrhage and grafting of dura thing are encapsulated in the thick-layer of connective tissue.To studies show that of the metathetical theme of suitable dura mater, early stage graft absorbed the formation coupling with endogenous new dura mater (neodura) in the past, was the tranquil principal element that merges with permanent dura mater of indication.

In the past, already several autologous tissues were used as the dura mater succedaneum.In 1911, Kostling formed the grafting of dura thing with patient's hernical sac.Kostling，W.，Med Wochenschr，58，1042(1911)。Other autologous tissues of fascia lata and periosteum lobe had been used such as temporal fascia since then already.The endogenous bigger nethike embrane of usefulness such as Barrow has successfully been rebuild big dura mater damage.Barrow etc., J Neurosurg.60; 305-1 (1987).The advantage of autograft is not have the danger of pathogen propagation or tissue rejection.But, extra taking-up tissue has increased operation wound, and may prolong the time of complicated operation process.

For many years already people's corpse dura mater of preserving is used as the dura mater succedaneum routinely, was used for people experimenter's dura mater displacement.Described preparation is made up of connective fiber, and described fiber is interlacing as the dura mater of health self.After people's corpse dura mater was used to neurosurgery, they were considered to form liquid-tight closure, were similar to the dura mater of health self, and were finally replaced by the tissue of health self in long-time degradation process.Material from corpse is by lyophilization (lyophilizing) and γ sterilization (Lyodura, B.Braun MelsungenAktiengesellschaft, Melsungen, Germany) or passes through multistage chemical process (Tutoplast ^Process, Tutoplast ^Dura, Tutogen Medical GmbH, Neunkirchen am Brand, Germany) preserve.But, people's corpse grafting of dura thing is relevant with the greater risk of carrying virus and Protein virus always, and described virus and Protein virus may cause the spongy encephalitis of terrorise's disease (Creutzfeldt-Jakob disease or GerstmannStreussler syndrome).Owing to after implanting people's dura mater, a large amount of death incidents occurred, so be restricted or forbid in a lot of uses of state household corpse grafting of dura things.

Also people's fascia lata and pericardium preparation are used as the dura mater substitute materials, it has the danger of the propagation infectious agent littler than people's corpse dura mater.Although this preparation has lower risk of disease transmission, they absorb slowly again, need the time of several months or several years, and this might cause the formation of cicatrix and the encapsulation of dura mater substitute materials.

The dura mater succedaneum also derives from inhuman source, as isolating cattle or pig collagen from skin or tendon and bovine pericardium tissue.Similar with the deutero-source of people, some cattle dura mater succedaneums are considered to always and can spread disease to the patient who accepts the grafting of dura thing, i.e. mad cow disease (BSE).But use the deutero-dura mater succedaneum of pig to cause adhesion with lower floor's cerebral tissue.

Narotam etc., U.S.5,997,895 disclose the dura mater succedaneum from the xenogenesis collagen of handling, and it is porous collagen sponge, felted or form of film.Processing to collagen makes virus and Protein virus pollute inactivation, thereby described succedaneum can not comprise the virus and the Protein virus of infection amount.According to open, the porosity of dura mater succedaneum is to allow vascular, cell and meningeal tissue infiltration dura mater succedaneum necessary.But, in clinical practice, the use of existing porous material also has shortcoming, because shape stability and main impenetrable liquid can not be always guaranteed.Narotam etc. also disclose the dura mater succedaneum, it be two or more collagen sponge, felted or form of film interlayer, wherein, at least a form has enough holes, so that meningeal tissue is inwardly grown.

Can also can be used for clinical application by resorbent polyester, but the shortcoming that has low elasticity and slowly degrade.Under special occasions, these implants may cause the wound healing problem, and may increase infection.

Already will be by being used as the dura mater succedaneum such as the metal of gold, platinum, silver, nickel, tantalum or steel or such as thin slice or lamella that the polymer of polytetrafluoroethylene (PTFE) or other polyester is formed.But these succedaneums can not be absorbed by the patient, but are encapsulated in the tough layer of connective tissue, and stay in the patient body throughout one's life as foreign body, and can not replaced by the structure of health self.The excessive risk that this may cause microorganism to be grown in endoporus is because the loose structure of PTFE leaf membrane makes this growth of microorganism can not obtain the control of the defense mechanism of health self.

Product based on collagen becomes more and more popular.Can have the structure of high connective tissue composition with the chemical method modification,, thereby have only acellular no antigenic collagen scaffold to preserve as pericardium or corium.Can obtain the complete product of forming by the synthetic material of collagen fibril or collagen coating.Under these two kinds of occasions, the collagen fiber network all plays a part the substrate of the endogenous connective tissue of growth.

Chaplin etc. have tested product (XenoDerm, the Lifecell Corp that obtains from guinea pig skin in animal model.，The Woodlands，TX)。Neurosurgery，45：2，320-7(August 1999)。Tester is from the body pericranium.In described production process, described epidermis, all cells composition and other potential antigenicities or infectiousness composition are removed by chemical method.Keep the collagen fiber and the structural system of skin constant.Reported soon to exist under the situation of slight cell effect, in the dura mater around described product can be incorporated into.Fibroblast preferably observes at implant site invasion property.Graft and original dura mater are considered to almost can't distinguish when research finishes after 6 months by a definite date operation.

According to above result, Warren etc. (2000) have studied and have used AlloDerm ^(LifeCellCorp., The Woodlands TX) carry out the dura mater displacement on people experimenter.Neurosurgery46(6)：1391-96(2000)。200 patients have received AlloDerm grafting of dura thing in this research.This material obtains from the human dermis.It is identical with XenoDerm that its production method is considered to, and produced acellular collagen bio-matrix, and it is that no main histocompatibility complex (MHC) is antigenic.There are 7 to develop postoperative complication among 200 patients,, still, do not have an example to be in the news because graft self causes as infecting and cerebrospinal fluid (CSF) fistula.Surgical repair is considered to show that none is at development adhesion of grafting of dura position or rejection among these patients.It is closely similar with dura mater on every side that described material is considered to when macroscopy.The data that study for a long period of time that also do not have at present this product.

Bovine pericardium (the Tutopatch that the γ that Filippi etc. (2001) preserve solvent sterilizes ^, Tutogen Medical GmbH, Neunkirchen, Germany) be used for carrying out the metathetical experiment of dura mater 32 experimenters.Filippi etc., Neurosurg.Rev, 24:103-107 (2001).Except a patient, process all is considered to eventless after all patients' the operation, and a described patient dies from the heart reason soon after operation.Described graft be said to be be convenient to handle, durable and cheaply.Still the data that study for a long period of time of not getting rid of possible late complication.

The collagen product is suitable as biomaterial and depends on multiple factor: chemotaxis interacts, and wherein, they have promoted endotheliocyte and fibroblastic rapid osmotic, and the latter produces and deposited new collagen fiber conversely again; Simultaneous limited lymphocyte inflammatory reaction in the structure has promoted the absorption of collagen bio-matrix around.Collagen also has haemostatic properties, and this characteristic is used to therapeutic purposes.Platelet itself is deposited on the collagen structure, decomposes, and discharges thrombin in this way, and this factor can promote fibrin to form in conjunction with blood plasma factor.

Known dura mater substitute materials and the relevant method of using described material, can not provide liquid-tight, can resorbent alternative dura mater, described alternative dura mater can be avoided encapsulationization, dura mater cicatrization or stick on the cerebral tissue, and, also have the lower risk of propagating microorganism, virus and Protein virus, described microorganism, virus or Protein virus may cause spongy encephalitis or other diseases.The displacement of ideal dura mater should not can cause the immune defence reaction, perhaps causes inflammation and must be nontoxic.It should be absorbed apace, and meanwhile makes and can form the connective tissue structure, thereby develops endogenous new dura mater.In this course, described graft should not can and cerebral tissue or bone adhesion or fusion.Described material should can anti tear, keeps its shape, and stops the cerebrospinal fluid infiltration.Described displacement dura mater also should be volume and dimensionally stable, and wherein, after implanting, it can resist expands or contraction.Other important criterion are virus and Protein virus safety, user friendliness and economic production cost.

Summary of the invention

Therefore, in various aspects of the present invention, provide displacement dura mater material, this material can be resorbent, liquid-tight, elasticity, volume and dimensionally stable, and the outstanding security feature relevant with the risk of pathophoresis is provided.

Therefore, say simply, the present invention relates to be used for repair and/or the method for regeneration dura mater tissue mammal.Allow described dura mater tissue contact with the horse collagen thin slice of the bio-matrix that comprises collagen fibril.Described horse collagen thin slice forms by a kind of like this method, wherein, makes the suspension precipitation of collagen fibril, so that form the collagen fibril thin slice, and wherein, described collagen fibril is not by chemical reagent or by crosslinking with radiation.

On the other hand, the present invention relates to be used to repair the method for mammal dura mater tissue, the horse collagen thin slice that comprises the atresia substantially of the bio-matrix that the non-natural that allows the dura mater tissue and comprise collagen fibril exists contacts, wherein, described horse collagen thin slice is become to be grouped into by acellular basically, and wherein, described collagen fibril is not by chemical reagent or crosslinking with radiation.

On the other hand, the present invention relates to be used for repair and/or the method for regeneration dura mater tissue mammal, comprise and allow the dura mater tissue contact with the horse collagen thin slice of the atresia of forming by the collagen bio-matrix basically substantially, wherein, described collagen bio-matrix is not by chemical reagent or crosslinking with radiation.

Other aspects and features of the present invention partly are conspicuous, and part will be pointed out hereinafter.

The accompanying drawing summary

Fig. 1 is SEM (scanning electron microscope) photo that shows exsiccant horse collagen sheet surface.Clearly show that collagen fibril.From photo, can obviously find out described surface imporosity substantially.

Fig. 2 A and 2B are the photos of taking under ESEM (environmental scanning electron microscope art) condition, this means it is approaching natural condition in moist slightly atmosphere, show the upper surface that looks from horse collagen foil side.Can obviously find out imporosity substantially from the photo.

Fig. 3 A and 3B are the photos of taking under the ESEM condition, show the lower surface of horse collagen thin slice.Collagen fibril as shown in Figure 3A.From photo, can obviously find out described surface imporosity substantially.

Fig. 4 is the SEM photo of the horse collagen sheet surface of expression hydration.In Fig. 4, clearly show that collagen fibril.From photo, can obviously find out described surface imporosity substantially.

Fig. 5 A, 5B and 5C are the photos of taking down in ESEM condition (humid atmosphere), show the cross section of horse collagen thin slice.Similar shown in the described material is in a lamination layer that very closely is deposited in together.Gap between the collagen layer has been shown in photo.

Fig. 6 A and 6B are the SEM photos of the exsiccant horse collagen thin slice cross section of expression.Gap between multilamellar collagen and the collagen layer has been shown in photo.

Fig. 7 is illustrated in to insert the horse collagen thin slice photo of the interior looks of operation of left side dura mater defective afterwards, and dura mater edge is on every side covered by blood clot.

Fig. 8 is the dye photo of micro-view (frontal section) of the Trichrom of expression operative site, shows cortex construction and (is positioned at the horse collagen thin slice on right side with two kinds of grafts; Be positioned at the Tutoplast in left side ^Dura) dura mater of Fu Gaiing (8X amplification).

Fig. 9 is illustrated in the photo of the grafting of dura thing in eight weeks afterwards of performing the operation.In the left side, Tutoplast ^Dura corpse grafting of dura thing looks and does not change to have apparent edge.Can see the remainder of the thin connective tissue membrane that covers graft.On the right side, horse collagen thin slice bio-matrix graft looks and has been entirely integrated in the dura mater on every side.Dark spots is to be caused by the little blood clot residue in the new dura mater.

Figure 10 is the expression operation photo of the macroscopy looks of corticipetal horse collagen thin slice bio-matrix graft eight Zhou Dynasty afterwards.Described graft looks smooth, can move, and in the dura mater around being entirely integrated into.Can't see the cortex damage.

Figure 11 is expression operation corticipetal Tutoplast eight Zhou Dynasty afterwards ^The photo of the macroscopy looks of Dura corpse grafting of dura thing.It is and even that described graft looks smooth, but the sign that graft is integrated do not occur.There is not the cortex cerebral meningeal adhesions.

Figure 12 is the expression operation photo of two all two multinuclear giant cells afterwards, and described cell has the interior fragment of the cell (hematoxylin (hematoxillin)-eosin (HE) dyeing of horse collagen thin slice bio-matrix graft; The 800X amplification).

Figure 13 is illustrated in fibroblast and the cytophagous photo that horse collagen thin slice bio-matrix graft has been permeated in operation afterwards all around.Can also see and have erythrocytic new capillary tube (neocapillary) (HE dyeing, 600X amplification).

Figure 14 is the segmental photo (HE dyeing, 600X amplification) of the horse collagen thin slice bio-matrix graft that surrounded by phagocyte and gentle lymphocyte inflammation around after the expression operation.

Figure 15 is Tutoplast around the expression operation afterwards ^The photo of Dura corpse dura mater.Described corpse dura mater shows minimum Premeabilisation of cells or graft is reinvented sign.On graft and below found fine and close lymphocyte inflammatory reaction (HE dyeing, 250X amplification).

Figure 16 is the operation photos of the micro-looks of eight all new dura maters afterwards, shows the residue layer (Trichrom dyeing, 150X amplification) of collagen fiber, fibroblast and the horse collagen thin slice bio-matrix graft of new formation.

Figure 17 is photos of implanting the micro-looks of new dura maters of 16 weeks after the horse collagen thin slice, shows fine and close collagen fiber and the new capillary tube that forms, and wherein has been full of erythrocyte (van Gieson dyeing, 200X amplification).

Figure 18 is the figure of the test set of the expression impermeability, tensile strength and the elasticity/flexibility that are used to measure displacement dura mater material.The protrusion degree of the described material that measurement is obtained by specific water-column is so that determine the elasticity/flexibility level.Measure the amount of pressurization, so that measure impermeability by the water of described test material.

Figure 19 is the hydrostatic pressure (water-column) that the is illustrated in increase collagen thin slice (collagen content: 5.6mg/cm of getting down from horse ²) the curve chart of protrusion degree.

Figure 20 is a collagen thin slice (collagen content: 4mg/cm under the hydrostatic pressure (water-column) that is illustrated in increase ²) the curve chart of protrusion degree.

Figure 21 is the curve chart of the protrusion degree of DuraGen under the hydrostatic pressure (water-column) that is illustrated in increase.DuraGen is at 200cm H ₂Break under the O hydrostatic pressure.

Figure 22 is the curve chart of the protrusion degree of several dura mater replacement product under the hydrostatic pressure (water-column) that is illustrated in increase.

Figure 23 is under the hydrostatic pressure (water-column) that is illustrated in increase, horse collagen thin slice (collagen content: 5.6mg/cm ²) the protrusion degree and the curve chart of the forfeiture of water.

Figure 24 is under the hydrostatic pressure (water-column) that is illustrated in increase, collagen thin slice (collagen content: 4mg/cm ²) the protrusion degree and the curve chart of the forfeiture of water.

Figure 25 is under the hydrostatic pressure (water-column) of expression increase, the curve chart of the protrusion degree of DuraGen and the forfeiture of water.

Figure 26 is under the hydrostatic pressure (water-column) of expression increase, the curve chart of the protrusion degree of some kinds of dura mater replacement product and the forfeiture of water.

Figure 27 is the curve chart of the tear strength/limit tension of several collagen implants of expression.Sample E is a horse collagen thin slice.

Detailed description of preferred embodiments

Be surprised to find according to the present invention, the thin slice of the atresia of being made up of the horse collagen fibril in the bio-matrix that non-natural exists substantially can be used as effectively can resorbent dura mater succedaneum, to be used for mammiferous dura mater reparation, regeneration and recovery, described mammal comprises people, laboratory animal etc.Horse collagen thin slice of the present invention is liquid-tight, and the high security feature of the risk that prevents that virus or Protein virus from propagating is provided.In addition, described horse collagen thin slice is actually pliability and elastic, has kept high tensile strength simultaneously.This thin slice below is referred to as " horse collagen thin slice ", and is when implanting, consistent with the key property of human dura mater.Described horse collagen thin slice plays a part the support of bio-matrix, is used for the inside growth of cells in vivo, and is replaced by new dura mater in regeneration and recovery period.

In one embodiment, described horse collagen thin slice bio-matrix is made up of the connective tissue protein that is made up of collagen fibril basically.Preferably, described horse collagen thin slice bio-matrix is made up of connective tissue protein, and described connective tissue protein is made up of collagen fibril.More preferably, described horse collagen thin slice bio-matrix is made up of connective tissue protein, and described connective tissue protein is made up of the type i collagen fibril.

Except being made up of collagen fibril, described horse collagen thin slice can also comprise excipient, antiseptic, somatomedin or help the pliability and the elastic additive of final products.

Horse collagen thin slice

Horse collagen thin slice of the present invention is the bio-matrix of the collagen fibril handled, has removed cell component by processing, and has formed the collagen fibril lamella.

Be used for the naturally occurring multilamellar collagem membrane of horse collagen thin slice right and wrong of one embodiment of this invention, its collagen fibril by a plurality of multi-direction mutual windings is formed.Figure 1 illustrates the figure of exsiccant horse collagen thin slice.The surface of described microphotograph (SEM) expression horse collagen thin slice wherein, is embedded with collagen fibril.Can see the photo of the horse collagen thin slice upper surface under ESEM (environmental scanning electron microscope art) condition in Fig. 2 A-2B, wherein, Chao Shi atmosphere provides approaching natural condition slightly.Described collagen fibril can be seen on described surface.Described surface looks to be slick and atresia substantially.The photo (ESEM) of horse collagen thin slice lower surface is provided in Fig. 3 A and 3B.Described lower surface photo also shows the imporosity substantially of horse collagen thin slice.

Before the described horse collagen thin slice of use was repaired mammiferous dura mater tissue, described exsiccant horse collagen sheeting can be hydration.Fig. 4 is the SEM photo on surface of the horse collagen thin slice of expression hydration, wherein, clearly show that collagen fibril.Described as can be seen surface imporosity substantially from the photo.

Even the collagen fiber of unique orientation also mainly are responsible for impenetrable liquid in the two-dimensional directional under high hydrostatic pressure in described multilamellar, and provide and had elastomeric high intensity.Because a plurality of parallel-oriented thin collagen fibril layer of horse collagen thin slice, so this material is fit to the interim dura mater that replaces health self, be used for after implanting, covering defective, so that obtain the closure that liquid-tight cerebrospinal fluid leaks, and provide for the ingrown bio-matrix support of cell, to be used to form new dura mater.This specific character is important in wound healing process, because it has reduced the risk that the patient is developed the cerebrospinal fluid gusher situation.

Horse collagen flake structure and absorb feature again

Described horse collagen thin slice is that its mammal of implanting can be resorbent.It is believed that the structure by horse collagen thin slice can strengthen this characteristic.Be used to produce the method for horse collagen thin slice, formed the lamination of collagen fibril.Between each layer is the gap, and patient's cell and vascular system can be moved in these gaps, and form new dura mater tissue.

Each layer collagen fibril is atresia substantially.A few hole that may exist is normally separated from one another, and can not be connected to each other by the multilamellar collagen fibril.Multiple structure of the present invention has strengthened the liquid-tight feature of horse collagen thin slice.Electron scanning micrograph shown in Fig. 1-4 shows the character of the atresia of horse collagen thin slice.

Although horse collagen thin slice is an atresia substantially, between the collagen fibril layer, there is the gap.Can find out described gap and stratiform feature easily in Fig. 5 A, 5B and 5C, described figure is the cross-sectional picture of horse collagen thin slice under ESEM condition (humid atmosphere).Fig. 6 A and 6B are the SEM photos of exsiccant horse collagen thin slice.Therefore, horse collagen thin slice is similar to a stacker and opens, and wherein, each layer paper is to be slick and atresia substantially, has the gap between each layer paper.Under its dried forms (Fig. 6 A and 6B), described gap is more obvious.When observing horse collagen thin slice under the approaching natural condition in humid atmosphere slightly, described gap smaller.Fig. 5 A, 5B and 5C are the photos of the cross section of horse collagen thin slice in humid atmosphere, wherein, show the dwindling of gap of described horse collagen thin slice.

Except promoting liquid-tight characteristic, a plurality of parallel-oriented thin collagen fibril layer of horse collagen thin slice plays a part simultaneously for the ingrown bio-matrix support of cell, so that from the beginning make up the dura mater of health self.In the past, generally believed that the porous support structure was that autonomous tissue of promotion and vascular system inwardly grow in the displacement dura mater tissue necessary.Be surprised to find already, the layer structure of the atresia of horse collagen thin slice can promote cell, vascular system inwardly to grow, and promote to pass horse collagen thin slice and in the gap that is present between its multilamellar, form new collagen structure, thereby form new dura mater, it is implanting the general layer structure that has natural dura mater within several weeks.As being further described hereinafter with among the embodiment 1, the inside growth of cell, vascular system, and new collagen structure is so extensively in several weeks after operation is difficult to dura mater tissue division with the patient who existed in the past to such an extent as to new dura mater becomes.In about 4-8 week after operation, the cell tissue of meningocyte is about 40%-70%.After about 16 weeks, described graft is organized fully (100%).

Zoopery has shown the quick Premeabilisation of cells at multilamellar horse collagen slice region.From histology's angle, after implanting 14 days, observed the intensive infiltration of lymphocyte, macrophage and fibroblast to the collagen bio-matrix.On graft, formed capillary tube subsequently.Horse collagen thin slice that occurs owing to the new life of collagen fiber and the successive transformation between the dura mater on every side are conspicuous.After 4 weeks only, described horse collagen thin slice is replaced by the loose structure organization of health self with regard to part.After 24 weeks, the new dura mater-sample connective tissue structure of the new formation of the horse collagen thin slice of the patient's who exists before being difficult to distinguish the dura mater and the implantation in substitutional defect zone.

Pathophoresis/immunoreation

The remarkable advantage that uses horse collagen thin slice of the present invention is to have the obvious lower risk of disease transmission to its patient who implants.Use acid (for example, hydrochloric acid, acetic acid etc.) in process of production, and alkali, as the naoh treatment collagen fibril so that produce such horse collagen thin slice, this can be advantageously with antibacterial, virus and the prion inactivation that may exist or reduce its infection level.Way with hydrochloric acid, sodium hydroxide, ethylene oxide processing biomaterials such as (ETO) had obtained the approval of government organs already, as the method that makes Protein virus and virally inactivated permission in the management of medicine and biomaterial.In some management, described processing can reduce the management expectancy of measuring horse collagen thin slice in batch.Therefore, the processing to collagen fibril has improved Product Safety in process of production, and has reduced the risk of pathophoresis being given the patient.

Having carried out the material of the horse of above-mentioned production process processing does not already find and can give the patient with any pathogen propagation.Therefore, except production process, based on the danger that the utilization of the collagen of horse has also avoided propagating spongy encephalitis, this disease was relevant with people's corpse succedaneum always in the past.Use comes from the collagen in source, Malaysia, for example comes from the collagen of horse heel string, has avoided the risk of propagation Transmissible spongiform encephalopathy (TSE), and this disease is known as mad cow disease (BSE) or pruritus disease again.The propagation of this disease always with use the biologic material that obtains from the ruminant source relevant (for example, from cattle, goat, sheep etc. biomaterial).

Horse collagen thin slice of the present invention has reduced extraly and has caused immunoreactive risk, and wherein, described collagen is from the source, Malaysia and handled (for example, using enzyme).Reported already and will be used for organizing immunoreation do not occur the time period above 10 years of method of replacing (tissue of displacement except dura mater) based on the collagen of horse.

The inflammatory reaction that the collagen thin slice that derives from horse has also caused weakening.Compare with comprising dura mater succedaneum from such as the collagen in the source of people's fascia lata, owing to implant substitute the inflammatory cell that horse collagen thin slice causes number obviously still less.Compare with displacement dura mater apparatus from other sources, shorter equally by the persistent period of implanting the caused inflammatory process of horse collagen thin slice.Above-mentioned characteristic has significantly reduced the risk to the transplant rejection of horse collagen thin slice that is produced by patient's immune system, thereby has improved the success rate that needs the metathetical neurosurgery of dura mater.

Volume/dimensional stability

If the displacement dura mater obviously expands or shrinks when hydration, may have problems.Under some occasion, the porous collagen dura mater replacement product of prior art is tended to obvious contraction after hydration.Under this occasion, the displacement dura mater may be drawn it is connected the epidural stitching thread of patient, thereby causes to implant with to the damage from body dura mater and operative site.Other complication comprise if after implanting the displacement dura mater described displacement dura mater prolonged expansion, at the pressure of operative site generation, thereby can in adjacent nervous tissue, produce undesirable pressure.

The change in volume of horse collagen thin slice of the present invention is less or insignificant when hydration.Opposite with the porous replacement product, described horse collagen thin slice can keep its size and dimension substantially after hydration, have good shape stability,, and after implanting, can in brain, not produce expansion or contraction problem even after hydration, also can keep Biostatic.In case hydration and implantation, horse collagen thin slice just can not torn operation suture thread or destroy the degree that horse collagen thin slice is remained on the epidural fibrin adhesive ﹠ sealing of patient significantly expanding aspect area or the thickness or be retracted to.

In one embodiment, when complete hydration, the contraction or expansion of the area of exsiccant horse collagen thin slice can approximately-about 20% scope of 5%-in.In another embodiment, when complete hydration, the area of exsiccant horse collagen thin slice can approximately-about 10% scope of 5%-in.In another embodiment, when complete hydration, the area of exsiccant horse collagen thin slice approximately-5%-about 5%.In another embodiment, when complete hydration, the area increase of exsiccant horse collagen thin slice is no more than about 4%.

In one embodiment, when complete hydration, described horse collagen thin slice is increased to maximum about 4 times of its dry thickness.In another embodiment, when complete hydration, described horse collagen thin slice is increased to maximum about 3 times of its dry thickness.In another embodiment, when complete hydration, described horse collagen thin slice is increased to about 2 times of its dry thickness.

Based on skull, the thickness of adult's dura mater is the about 2.0mm of about 0.5mm-.The thickness of dura mater also can depend on patient's age and change, and wherein, estimates that baby and child's dura mater is organized generally thinner than adult.The thickness of horse collagen thin slice of the present invention can be prepared into the usable floor area and the patient of treatment and changing as required.

In one embodiment, the thickness of horse collagen thin slice of the present invention under its dried forms is the about 3.0mm of about 0.01mm-.In another embodiment, the thickness of described horse collagen thin slice is the about 2.0mm of about 0.02mm-.In another embodiment, the thickness of described horse collagen thin slice is the about 1.5mm of about 0.03mm-.In another embodiment, the thickness of described horse collagen thin slice is the about 1mm of about 0.05mm-.In another embodiment, the thickness of described horse collagen thin slice is about 1.0mm or following.

The dry weight of collagen thin slice depends on its ideal thickness.In one embodiment, the dry weight of horse collagen thin slice is about 1mg/cm ²-about 50mg/cm ²In another embodiment, the dry weight of horse collagen thin slice is about 1.5mg/cm ²-about 30mg/cm ²In another embodiment, the dry weight of horse collagen thin slice is about 2mg/cm ²-about 20mg/cm ²In another embodiment, the dry weight of horse collagen thin slice is about 2.5mg/cm ²-about 15mg/cm ²In another embodiment, the dry weight of horse collagen thin slice is about 3mg/cm ²-about 10mg/cm ²

In one embodiment, the weight of horse collagen thin slice is increased to maximum about 15 times of its dry weight after hydration.In another embodiment, the weight of horse collagen thin slice is increased to maximum about 10 times of its dry weight after hydration.In another embodiment, the weight of horse collagen thin slice is increased to maximum about 7 times of its dry weight after hydration.In another embodiment, the weight of horse collagen thin slice is increased to maximum about 5 times of its drying regime after hydration.

For as suitable dura mater displacement succedaneum,, on the contrary, should have quite high stability/tensile strength even the dura mater succedaneum of implantation also should not can when hydration become shaky.Horse collagen thin slice of the present invention advantageously has high tensile strength, this can improve and be supported on operation use in to the processing of horse collagen thin slice, and be provided at the mechanical stability of the increase after implanting.Control experiment is provided in the following embodiments, and wherein, the tensile strength of described horse collagen thin slice and porous collagen preparation (for example, collagen foam) are compared better.In addition, increase the thickness of horse collagen thin slice, can significantly improve tensile strength.

The tendency that horse collagen sheeting is torn under applied pressure can be measured as its " ultimate elongation load " or " limit tension ", below is referred to as " limit tension ".The limit tension of horse collagen thin slice can be by exerting pressure to a horse collagen thin slice with specific width, and measure and cause described horse collagen thin slice to destroy the amount of (for example, tear or break) institute's applied pressure and determine.Can carry out quantitatively limit tension in order to following equation:

Width=newton/cm-the bar of " limit tension "=applied force/horse collagen strip of foil.

In one embodiment, the limit tension of described horse collagen thin slice is about 30 newton of about 1-/cm-bar, about 15 newton of 1.5-/cm-bar preferably approximately, about 10 newton of 2-/cm-bar preferably approximately, more preferably about about 6 newton of 3-/cm-bar.

Although horse collagen thin slice of the present invention has high tensile strength, it also can keep elasticity and pliability when hydration.This feature makes described horse collagen thin slice be suitable for appearing at the anatomical conditions of implant site (for example, bending) best.

Under its hydration status, described horse collagen thin slice can easily move around operative site, and can carry out the shape of model for its defective of implanting best.In case implant, described horse collagen thin slice graft just keeps smooth and mobility.As time passes, cell and vascular system migration are by described horse collagen thin slice, and finally the new dura mater with the dura mater sample replaces it.With the tissue of dura mater cell generation cell after, described horse collagen thin slice can not stick on nerve, brain, skull or the spinal tissues.

The preparation of sections of horse collagen

Horse collagen thin slice of the present invention can be by the suspension preparation of in check drying means with the high molecular collagen fibril.The fractional precipitation of collagen fibril suspension be since raise evaporation of water and pH the time cause.Described in check drying means has produced the multiple structure of collagen thin slice, and this multiple structure can be implanted as people's dura mater succedaneum by neurosurgeon.Described multilamellar collagen flake structure provides multiple above-mentioned characteristic favourable in the dura mater succedaneum, and provides as the dura mater tissue of bio-matrix to live again.

In one embodiment, the method that is used to produce horse collagen thin slice of the present invention has been removed all cells composition of producing the horse collagen thin slice of collagen fibril, and described thin slice is become to be grouped into by acellular basically.

Utilize the method for setting up in the collagen chemistry, the tissue that will contain collagen is used as raw material to be used to prepare horse collagen thin slice of the present invention.In one embodiment, the horse tendon is used as raw material.In another embodiment, the horse heel string is used as raw material.

In one embodiment, described raw material, for example the horse heel string is at first pulverized, and with 1N naoh treatment at least 1 hour, and neutralizes with hydrochloric acid.Under the acid condition of pH 2, handle described collagen raw material.Used acid can be hydrochloric acid, acetic acid etc.Then, noncollagen protein and the intermolecular cross-linking key that is present in the described raw material carried out enzymatic degradation with pepsin, so that form the collagen suspension.

Described suspension then neutralizes.In one embodiment, described suspension the is neutralized about pH 8.0 of about pH 6.5-.In another embodiment, with the described suspension about pH 7.5 of about pH 6.9-that neutralizes.In another embodiment, with described suspension about pH 7 that neutralizes.

Described collagen suspension is carried out centrifugal, remove supernatant, and will precipitate and be suspended in again in about pH 2-4.5 acetic acid.Therefore from the collagen suspension, successfully removed noncollagen protein.

Can be according to required repetition above-mentioned steps, so that remove the noncollagen protein that is present in the remnants in the precipitation.

The surprising result of the production method of described horse collagen thin slice is that it is because by long-time, for example, 24 hours evaporation is specifically removed water and realized that the in check pH of the suspension of collagen in acetic acid raises.The specific rising of described pH has caused the precipitation of collagen fibril in the layer of two-dimensional directional of multi-direction winding, thereby has formed the multiple structure of described horse collagen thin slice.In one embodiment, described process is to carry out in drying oven under about 20 ℃-about 55 ℃ temperature, wherein removes devaporation with equipment, simultaneously acetic acid is carried out the steam neutralization.In another embodiment, described process is to carry out in temperature is the drying oven of about 30 ℃-about 45 ℃ temperature.

From the described horse collagen thin slice of described production process when detecting the dried forms that is considered to be in it in the time of can ignoring less than the further forfeiture of the further forfeiture of moisture or moisture.The water content of " dried forms " of horse collagen thin slice is generally the about 2%-about 18% that calculates by weight percentage.In " dried forms " of described horse collagen thin slice, exist high relatively residual water content to stop or limited the degeneration of the tropocollagen molecule that constitutes described horse collagen thin slice.

Said process is determining the precipitation of collagen fibril from suspension, and this is because the composition sedimentation when low pH value elevation process begins (fall out) with low solubility.This technology has caused the precipitation of collagen fibril during water evaporates and the rising of pH simultaneously.

In described precipitation process, along with described fibril is precipitated out from described solution, described collagen fibril can take place natural crosslinked, thereby forms the collagen thin slice.With (for example use chemical reagent or radiation, ionization or ultraviolet radiation) the crosslinked difference of collagen fibril, this may cause having increased soak time again, makes the natural crosslinked energy of collagen fibril promote soak time short when described horse collagen thin slice is implanted.Being used for the fibriilar natural crosslinked of collagen thin slice of the present invention can be undertaken by natural, physiology's sample loading mode.At first, this natural crosslinked be to realize by noncovalent interaction (for example, Van der Waals interact or dipole-dipole interaction) or by between the amino acid side chain of tropocollagen molecule, forming easy dissociated schiff bases key.The intermolecular cross-linking decision physics and the chemical stability of collagen.The committed step that collagen cross-linking forms depends on the Enzymatic transformation of lysine or hydroxylysine residue, and has produced aldehyde, allysine and 6-hydroxyallysine.Described aldehyde radical can spontaneously react with the active amino group, has caused comprising having (the formation of the schiff bases composition of unsettled aldol condensation product CH=N-) of unsettled aldimine key.Therefore, the fibril of product of the present invention can dissociate by using such as faintly acid processing.By crosslinked can the detection of using that chemical cross-linking agent produces according to the existence of the crosslink part of stable covalent cross-linking.Generally, this purpose is by using Schiff-base reagent (for example, glutaraldehyde) to form the schiff base reaction product, resets by Amadori then or reducing condition stablizes that described key realizes.In addition, collagen can be undertaken crosslinked by various difunctional carbodiimide reagents.By crosslinked can the detection by the existence of covalent bond stable between the collagen fibril of using radiation to produce, this covalent bond is to produce by the free radical reaction partly that produces between radiation era.On the other hand, the fibril in the product of the present invention is substantially with any stable covalently cross-linked, and chemistry of no use or method of radiating are handled.Therefore, any combination in the product of the present invention between the fibril is non-covalent substantially or reverses easily, and do not stablize crosslinked.To be used for the collagen fibril chemical crosslinking at the dura mater succedaneum such as the chemical reagent cyanogen ammonia, glutaraldehyde, formaldehyde, acrylamide, carbodiimide diketone, two imino-esters (diimidates), the bisacrylamide etc. already in the past.But, use this chemical reagent, may cause and the unexpected relevant risk of toxicity of remnant chemical reagents that contacts in the dura mater succedaneum of cerebral tissue.Therefore, this intermediate processing has been avoided the risk of toxicity of cross-linking chemistry reagent, and with the relevant long soak time again of chemical reagent or crosslinking with radiation collagen fibril.

Resulting exsiccant, sedimentary collagen compositions has formed the horse collagen thin slice of being made up of high molecular multilamellar collagem membrane, and described collagem membrane is made up of the collagen fibril of a plurality of multi-direction natural windings.Described horse collagen thin slice mainly comprises the type i collagen in gap.Described horse collagen thin slice is atresia basically, and mainly is liquid-tight.Can on described product, carry out immunodiffusion test, so that guarantee not exist heterologous protein.

Be used for producing be used for described horse collagen thin slice of the present invention said method also by ResorbaWundversorgung GmbH ﹠amp; Co.KG, Nuremberg, Germany is used for producing by Baxter AG, Vienna, the commercial collagen thin slice of selling of Austria.Described commercialization thin slice is indicated to can be used as hemorrhage, as interim tissue substitute, is used to cover wound, and as fibrin sealer carrier mass.

The thickness that is used for horse collagen thin slice of the present invention can change according to the needs of application-specific.For example, when reparation department of pediatrics dura mater is organized, can use thin horse collagen thin slice, and when reparation adult dura mater is organized, can use thicker horse collagen thin slice.

By the raw-material amount that change is used to produce specific dimensions horse collagen thin slice, can control the thickness of horse collagen thin slice.

With ethylene oxide (ETO) or similar sterilizing gas or described horse collagen thin slice is carried out gaseous sterilization by radiation.

Adherence method

Before using, can make the hydration of exsiccant horse collagen thin slice, for example, hydration in normal saline.In one embodiment, described normal saline comprises 0.9% sodium chloride solution.In another embodiment, described horse collagen thin slice is at excipient or contains hydration in the solution of medicine.Make the needed time span of described horse collagen thin slice hydration relevant with the thickness of described thin slice.Making the hydration of described horse collagen thin slice, is uniform on whole area up to its thickness.In one embodiment, in normal saline, make about 5 seconds of described horse collagen thin slice hydration-about 1 hour.In another embodiment, in normal saline, make about 5 seconds of described horse collagen thin slice hydration-about 30 minutes.In another embodiment, in normal saline, make about 5 seconds of described horse collagen thin slice hydration-about 20 minutes.In another embodiment, in normal saline, make about 5 seconds of described horse collagen thin slice hydration-about 10 minutes.In another embodiment, in normal saline, make about 1 minute of described horse collagen thin slice hydration-about 6 minutes.In another embodiment, in normal saline, make about 5 minutes of described horse collagen thin slice hydration.In another embodiment, before implanting, described horse collagen thin slice is not carried out hydration.

Described horse collagen thin slice can be by the surgical technic determined attached on patient's dura mater, for example, and by fibrin sealer, tissue glue, operation suture thread or by the pressure fitted surgical technic.In addition, can utilize natural captivation between horse collagen thin slice and the dura mater tissue with described horse collagen thin slice attached to the dura mater tissue, and need not use any sealer, glue, stitching thread or pressure fitted technology.In case hydration, described horse collagen thin slice can be cut into bigger than the epidural surgical openings of patient slightly.Therefore, with described horse collagen thin slice slightly imbrication on its accompanying patient's dura mater.In one embodiment, the size of the horse collagen thin slice of hydration and the about 1cm of the overlapping about 0.5cm-of dura mater.Eclipsed amount can change according to the preference and the technical ability of neurosurgeon.

In one embodiment, according to the interaction of well-known collagen and fibrin, can use the fibrin sealer that can be used for the neurological purposes that is given the ratification with described horse collagen thin slice attached on the dura mater.The example that goes through to can be used for the fibrin sealer of neurological purposes comprises Tissucol and Tisseel fibrin sealer (Baxter AG, Vienna, Austria).In addition, can also use the tissue glue that is approved for the neurological purposes.Fibrin sealer or tissue glue can use with the form around the continuous lines of the horse collagen sheet segment of imbrication dura mater, so that form liquid-tight sealing.As indicated above, liquid-tight sealing is favourable, because it has been avoided and cerebrospinal fluid is lost relevant complication, as cerebrospinal fluid gusher.

In another embodiment, described horse collagen thin slice has produced liquid-tight sealing by the line of successive fibrin sealer or tissue glue and when the dura mater of body adheres to.

In another embodiment, the described horse collagen thin slice of imbrication dura mater can be with the fibrin sealer of mottled use or tissue glue attached on the dura mater.

In another embodiment, in a single day described horse collagen thin slice is placed on ideal implant site, just by operation stitching attached on the dura mater.Although this embodiment can be used for described horse collagen thin slice attached to the patient on the dura mater of body, described stitching thread may cause ramose pipe, the latter may cause fistula again conversely, and causes cerebrospinal fluid to leak.If sew up described horse collagen thin slice, must use no tension force suturing skill, so that avoid tearing described thin slice.Recommend the sealing stitching thread, for example, use the fibrin sealer.

In another embodiment, described horse collagen thin slice is according to pressure fitted technological orientation known in the art and implantation.In this technology, described horse collagen thin slice is placed on the implant site that needs, and remains on the appropriate location by the natural internal pressure that is present in skull or the spinal column.Therefore, graft still remains on the appropriate location under the situation of not using operation suture thread, fibrin sealer or tissue glue.

In another embodiment, under the situation of not using any sealer, glue, stitching thread or pressure fitted technology, described horse collagen thin slice is positioned and implants.In this technology, described horse collagen thin slice is placed on the implant site that needs, and remains on the appropriate location by natural captivation or the adhesive force between described horse collagen thin slice and the dura mater tissue.

Horse collagen thin slice of the present invention can be as displacement grafting of dura thing to repair because other operation processs of patient's dura mater maybe may be destroyed or penetrate to congenital situation, birth defect, disease, damage, tumor resection, maybe need to repair any other situation of dura mater and the human dura mater tissue that causes.Described horse collagen thin slice also can be used for repairing other mammiferous dura mater tissues, includes, but are not limited to sheep, monkey, horse, laboratory animal or other mammals.Described horse collagen thin slice can be used for repairing skull or along the dura mater tissue of spinal column.

The invention still further relates to and comprise horse collagen thin slice and instruct its preparation and as the test kit of description of displacement dura mater.

Contraindication

Known have allergic patient to horse or horse product and forbid accepting horse collagen thin slice.

Other contraindications are carried out radiocurable patient after can being included in operation soon.For example, accepting radiocurable patient soon after cerebral tumor resection operation, is not the good candidate who receives horse collagen thin slice of the present invention.Described radiotherapy may delay or suppress the growth of new dura mater, and described new dura mater is made up of quick splitted cell, and wherein, described horse collagen thin slice is absorbed again.Under this occasion, can not resorbent displacement dura mater, may be more suitable for as Teflon.But, skilled surgeon can recognize that needs can not resorbent metathetical treatment.

Definition

The bio-matrix (being the substrate of biocompatible material) of " horse collagen thin slice " expression horse collagen fibril has carried out handling so that remove cell component to it, so that form the collagen fibril lamella.Term " horse collagen thin slice " does not comprise the compound foil of the collagen fibril lamella that is combined in the one or more atresias substantially on one or more porous collagen lamellas.

The mammiferous dura mater tissue of " dura mater tissue " expression from body.

Substrate or support that " bio-matrix that non-natural exists " expression is made, it comprises the collagen fibril that is formed by the following: (1) naturally occurring material (promptly, natural material), by certain way it was carried out handling or processing already, wherein make the collagen fibril that comprised in the described natural material already the natural alignment in collagen structure at natural material taken place to move or reset; Or the material (that is non-natural material) that handle with collagen fibril (2) or finished non-natural exists.For example, the bio-matrix of non-natural existence can be made with the raw material that comprises the collagen that has carried out machinery or chemical treatment (for example, pulverizing, chopping etc.).On the contrary, by handling in a certain way or collagen bio-matrix that the processing raw material the is made naturally occurring bio-matrix of right and wrong not, described mode keeps the structure of collagen scaffold (for example, epidermal tissue to be handled, so that remove cell component, keep naturally occurring collagen structure simultaneously).

Any hole that " atresia substantially " expression is present on the horse collagen thin slice is the collagen fibril sedimentary result who forms the collagen lamella, and they mainly are isolated from each other.Can interconnective hole not interconnective with the form of the thickness that passes horse collagen thin slice.The machine drilling that forms the hole on described horse collagen thin slice is not the hole.Preferably, look that the material of atresia can be seen the hole when using scanning electron microscope to check with the 1500x amplification substantially.

The following examples will further specify the present invention.

Embodiment 1

Present embodiment provides the assessment horse collagen thin slice that carries out with sheep to be used to the experimental result of repairing the dura mater tissue and being used for the regenerated well-formedness of dura mater as bio-matrix as the dura mater succedaneum.

Experimentize, so that assessment horse collagen thin slice is as the characteristic of skull dura mater succedaneum, as studying with the sheep model.Described horse collagen thin slice comprises the natural horse collagen fibril (5.6mg/cm of purification from the horse heel string of chopping ²) and do not comprise cell component.

Employed reference product is the people's corpse dura mater (Tutoplast that preserves ^Dura).Only use fibrin adhesive ﹠ (Tissucol Duo S Immuno, Baxter AG, Vienna, Austria) with these two kinds of products attached to the appropriate location.

Studied following project:

The macroscopy looks that these two kinds of grafts are integrated;

The reaction of adjacent organizational structure (inflammation, adhesion, fibrosis, necrosis); With

The Histological assessment that the tissue of integration process and connective tissue is carried out

The purge process that is used to produce horse collagen thin slice starts from handling described tendon raw material at least 1 hour with sodium hydroxide solution, neutralizes with hydrochloric acid then.Decompose described tendon with pepsin then.Consequent colloidal collagen precipitates with the fibril form.Produced horse collagen thin slice by dry and gaseous sterilization then, wherein every square centimeter of natural collagen fibril with 5.6mg.Do not add any other composition, and do not carry out crosslinked (promptly relating to chemical reagent or radiation) by manual method.The immunodiffusion test guarantees not exist foreign protein.

Material and method

Laboratory animal

This research is carried out with 25 adult sheep.Described sheep is miscegenation (mixed-breed) domestic animal that is used for agricultural.The average weight of these animals is 53.0kg when operation, and their mean age is 2 years old.All animals all are female.These animals remain in the animal fence of LuebeckMedical University, and described fence is equipped with roof structure and open-air fence.For these animals provide conventional mixed fodder.These animals are divided into five groups carry out histology experiment, and the different phase that graft is integrated characterizes (first group the-the 5th group).Every group time-to-live was 2,4,8,16 and 24 weeks.

The research product

The horse collagen thin slice of research is made (type i collagen that is mainly the gap) with natural horse collagen fibril.One square centimeter material comprises 5.6 milligrams of collagen fibrils, does not contain cell component.Tester (Tutoplast ^Dura, Tutogen Medical CmbH, Neunkirchen a.Brand, Germany) be people's corpse dura mater by organizing reduction method to preserve.

With fibrin adhesive ﹠, Tissucol Duo S is used for graft attached to dura mater.This biology, two component glue were by the syringe that human plasma protein fraction, fibrinogen, Hageman factor I, blood plasma fibronectin and aprotinin are housed that is pre-charged with thrombin is housed and another syringe that is pre-charged with of calcium chloride is formed.

Anesthesia

By with injection mixture intramuscular injection following compositions to the preceding medication that undergos surgery of described animal: xylazine hydrochloride (xylazine hydrochloride 2%, Bayer AG, Leverkusen, Germany), dosage: the 0.1mg/kg body weight, (S)-ketamine (ketamine S, Parke-Davis GmbH, Karlsruhe, Germany) dosage: 2mg/kg body weight and 0.5mg atropine, 1ml injection solution (Atropinsulfat Braun 0.5mg, B.Braun Melsungen AG, Melsungen, Germany).Insert vein and arterial in right ear.Use Propofol (Disoprivan 2%, AstraZeneca GmbH, Wedel, Germany) with the 1mg/kg body weight and be used for anesthesia.Described animal is carried out endotracheal intubation (I.D.7.0mm), and use 100% oxygen, so that the normal ventilation of control (normoventilation) (Sulla 808V anesthetic ventilator, Dr  ger, Luebeck, Germany).

Use Propofol, (S)-ketamine and sevoflurane so that keep equilibrated anesthesia.Oxygen fraction (Oxydig, Dr  ger, the Luebeck of pressure in intra-operative monitoring breath cycle and volume ratio, suction, Germany), the final carbohydrate concentration (Kapnodig that exhales, Dr  ger, Luebeck, Germany), electrocardiogram and invasion arteriotony.

Preoperative antibiotic prophylaxis

Before being about to undergo surgery, each animal intravenous receives the dosage (Basocef 2.0g, Curasan AG, Kleinostheim, Germany) of 2.0g cefazolin.By the long-acting product (depot product) of twice subcutaneous administration, (1.0ml comprises 100 000IU benzathine benzylpenicillin and 100 000IU dihydrostreptomycin sulfates to the Strepdipen-suspension after operation; Dosage 1.0ml/kg body weight) keeps four days antibiotic prophylaxis again.Described subcutaneous injection carries out and carried out once more after 48 hours after operation finishes at once.

Surgical technic

The animal of intubate is already placed with leftward position.Then head is turned to the right side, and remain on horizontal level on the operating-table by being clipped in.Then skull is thoroughly picked hair, skin is carried out defat, then sterilization with gasoline.Fixedly have the aseptic lamella that exposes the position opening that will undergo surgery, and cover whole animal with sterile hood.

Form the skin incision of 1.5cm in the intermediate line left side, and prolong about 6cm.Use bipolar tweezers to solidify hemorrhage from the scalp that peels off.Use drag hook, and the temporoparietal region of skull is exposed by shrinking and launching galea aponeurotica.Use mechanical awl to bore two holes (diameter 0.8cm) then, its at interval about 5cm.With the oblong basin on the skull between saw (Mikrotom, Aesculap, Melsungen, Germany) the removal boring.

By using bone wax to stop any hemorrhage from skull.On dura mater, cut the long otch of about 0.5cm with scalpel.Cut the oval dura mater sheet of about 3 * 2cm then along the edge of described bone with the dura mater shears.Pay special attention to so that do not damage arachnoidea.Use hemorrhage, TachoComb ^(Nycomed Austria GmbH, Linz, Austria) prevention is any hemorrhage from the dura mater blood vessel.

Cutting oval horse collagen thin slice then (is of a size of 3.5 * 2.5cm) to a certain size, and soaked 5 minutes in aseptic 0.9% saline.In order to seal described defective, described horse collagen thin slice is filled in along the whole edge of dura mater, and put and go up fibrin adhesive ﹠, so that hold it in certain position.Referring to Fig. 7.

Use two platelets (miniplate) (Bioplates, Codman, Norderstedt, Germany) to adhere to the skull dish again then.With the described medicated cap shape film of the stitching thread that can absorb (Vicryl 2.0) sealing, and with Ethilon 3.0 skin sutures.

With Tutoplast ^The Dura product is applied in the right side of skull in a similar manner.Referring to Fig. 8.These two wounds are finally all used and are sprayed dressing (Hansaplast Spr ü hpflaster, BeiersdorfAG, Hamburg, Germany) processing.

Mean operative time is 120 minutes.Is about 60 minutes from induced anesthesia to section average time that begins to perform the operation, and from stitching finish to the average time that anesthesia finishes be 5-10 minute.

Observe after the operation of animal

After pulling out intubate, described animal sent in about 30 minutes back to their fence.Make regular check on inflammation or the unusual symptom of neurological of these animals by surgeon, veterinary and animal care person.After operation, described animal put into open-air fence in eight days.

The animal mercy killing

The predetermined time-to-live kill animals in 2,4,8,16 and 24 weeks is so that sampling after operation.

Before slaughtering, carry out calm by 2% pair of described animal of xylazine hydrochloride of intramuscular injection 1mg/kg body weight.Buckle well electrocardiogram (ECG) monitor, and vein or arterial are put into right ear.Then by intravenous injection T-61 ^(Hoechst Roussel Vet, Somerville, New Jersey) slaughters degree of depth abirritative animal; The injection solution of 1ml comprises 0.2g embutramide, 0.05g mebezonium iodide and 0.005g tetracaine hydrochloride; Dosage is the 0.3ml/kg body weight).Monitor described process by the measurement of ECG and arteriotony.

Sampling

Pick the hair on the described animal head, and position by operation method is described.Forming diameter on the skin around two operative scars is the circular incision of about 9cm.Shrink galea aponeurotica,, and hole at the right side front region so that expose the large slice of skull lid.Take out the circular skull dish that is approximately 8cm with saw.Whole transplantation site is made up of bone, dura mater and brain essence, by entering this position with dissecting knife along the bone edge cuts.Then carefully with the osteodiastasis of dura mater and cerebral tissue and covering, and fixing in formalin, so that carry out Histological research.The diameter of described graft sample is approximately 7cm.

Histological method

Described graft sample is carried out macroscopy, and be divided into volume section (frontalslice).Prepare two operative sites simultaneously.Taking out thickness from each sample is five sections of about 2-3 μ m.

Utilize the assessment of standard colouring method to change, comprise with hematoxylin-eosin and assess cell component, with Elastica van Gieson assessment mesenchyme structure, with Trichrome assessment mesenchyme structure, and assess collagen fiber new life, and determine hemorrhage degree with the ferrum dyestuff

The result

In the operation and operation back process

Follow-up period all is eventless after anesthesia, operation and the operation of all animals except two animals.An animal is dead during induced anesthesia, and this is because refractory arrhythmia.The another animal after operation 14 days suddenly and unexpectedly dead, be without incident operation process afterwards before this always.The micrography that brain is carried out shows cortical necrosis widely, has the sign that cicatrix occurs.Therefore, causing dead most probable reason is the long-term cerebral ischemia that causes owing to the unknown cause of disease.

The operation internal hemorrhage seldom appears, this hemorrhage be slight, and under most of occasion from the little blood vessel of dura mater.Use has bipolar tweezers or the rapid control over bleeding of hemorrhage.

Follow up a case by regular visits to after operation in the process, there do not have an animal to show neurological to be unusual.Equally, there is not animal to show that inflammation, cerebrospinal fluid are leaked or the symptom of the wound healing that sustains damage.

Macroscopy

During taking out sample, following parameter is checked with quantitative from operative site:

The formation of adhesion between-skull and the dura mater;

-cerebrospinal fluid leaks and inflammatory activity;

The visible variation of-grafting of dura thing; With

Adhesion of-meninges cortex and cortical reaction.

Microscopy

In accordance with the following methods Histological section is carried out the systematicness assessment:

-inflammatory reaction of transplantation site is described and quantitative (transition region under epidural, the dura mater, between dura mater and graft);

The degree of the tissue of-graft connective tissue;

-antixenic degree;

Change (the inflammatory process of-subarachnoid space; Fibrosis is to open subarachnoid space); With

The change of-cortex (inflammation, necrosis).

Macroscopy and the result of Histological research

Below the described result of Histological research identical aspect the cell composition, and aspect intensity difference, described research is to use from the different volume section of same animals and on the same group all animals mutually to carry out.

The macroscopic evaluation that graft is integrated

After 2 time-of-week sections, take out skull, between the bone of fibrin adhesive ﹠ residue and covering, found the adhesion of the minimum of bilateral.This adhesion is unclamped easily.The sign that inflammation or cerebrospinal fluid ooze out does not all appear at any grafting of dura position.All be dispersed with mottled independently blood clot on two kinds of grafts, its diameter is several millimeters.

On left hemisphere, described horse collagen thin slice still limits to equally, and looks more weaker than the initial ground glass outward appearance transparency that becomes.With described dura mater carefully when cortex takes off, described collagen product keeps adhering to described dura mater edge.Have a few very slight adhesion, these adhesions are easy to unclamp and don't damage cortex.

With the naked eye look right Tutoplast in the hemisphere ^Dura does not change.When taking out described product, in graft-dura mater contact area, Tutoplast ^Dura separates on certain position.With dura mater when cortex takes off, adhesion under the arachnoidea is seldom arranged, but, be easy to these adhesions be unclamped with tweezers.

Around after the operation, the imbrication bone and below the edge of dura mater between have the adhesion of minority; These adhesions are because the residue of fibrin adhesive ﹠ causes.Described bone can unclamp from dura mater under the dilatory situation at an easy rate not having, thereby can not cause the damage to dura mater or graft.At the described horse collagen thin slice position that is positioned on the left hemisphere, the watershed area between dura mater and the graft is no longer clear and definite.Described graft becomes not too transparent with comparing in the past, and presents light red.The looks of described graft towards the surface of brain are uniform, slick and transportable.No longer there is adhesion under the dura mater.Can see a few blood clot residue.

With the naked eye look Tutoplast ^Dura does not change equally.Combination inadequate, that unclamp has easily been found in contact area between inspection graft and the dura mater.

In eight weeks after the operation, the transition region between dura mater on the left hemisphere and horse collagen thin slice graft no longer exists.Structural continuity in the meninges both sides is tangible.The zone of placing described horse collagen thin slice only appears on the film of attenuation slightly, and presents the light red outward appearance.Referring to Fig. 9 and 10.Be arranged in the Tutoplast of right hemisphere this moment ^The both sides of Dura graft are all covered by thin connective tissue membrane.Referring to Figure 11.Below eclipsed dura mater, the edge of graft is still high-visible.To Tutoplast ^Drawing that Dura is slight just is enough to make it to separate with preformed dura mater.

After 16 weeks, new dura mater is formed on described horse collagen thin slice position and further develops, and described dura mater and new dura mater undistinguishable almost.

Be positioned at the Tutoplast on the right hemisphere ^The connective tissue encapsulation of Dura graft becomes more clear.

After 24 time-of-week sections, the section of the section of two transplantation sites and last group does not have difference on macroscopy.

The microscopic evaluation that graft is integrated

Positive according to expectation, in two weeks after operation, the zone of inflammation change has widely appearred in described horse collagen thin slice graft site.Whole subarachnoid space is by the adhesion closure, and this adhesion is owing to a large amount of exudates from lymphocyte, the granulocyte of cutting apart and macrophage cause.On described graft, having very widely, some zone of inflammation exudate is tangible equally.Except lymphocyte and mononuclear cell composition, also have little GUSUIPIAN to have multinuclear giant cell and the reaction of foreign body accordingly.

Described horse collagen thin slice graft itself shows the invasion of the loose and inflammatory cell of homogeneous texture, is heart-shaped or widely.Referring to Figure 12.Under the minority occasion, there is the ischemic necrosis of (relevant) corticocerebral shallow list area with operation.

Tutoplast ^The Dura sample shows inflammatory reaction widely equally, particularly at subarachnoid space.Graft itself can not permeated by inflammatory cell, still, is identifiable at two ends inflammation lymphocyte and mononuclear cell and foreign body reaction.

Around after the time period, the inflammatory of described horse collagen thin slice transplantation site changes obvious decline has taken place, and still, still has lawn sample lymphocyte and mononuclear cell exudate.Referring to Figure 13 and 14.Multinuclear foreign body giant cell is more common, particularly at close GUSUIPIAN place.In initial horse collagen thin slice graft, can see a plurality of fibroblasts.In HE (hematoxylin-eosin-dyeing) section, the general homogeneous texture of graft be detect less than.Show collagen fiber new life widely with EVG (Elasticavon Gieson) and the former transplantation site of the painted sample of trichrome.Actual dura mater is successfully given way in the tissue of loose structure, and this tissue is made up of the new collagen fiber that form that show the inflammatory infiltration.Observed leptomeningeal inflammatory adhesion before no longer existing.There is subarachnoid space equally, thereby produces breach.Pia mater encephali continues to show the little tuberosity of lymphocyte-mononuclear cell in the certain position infiltration.

At Tutoplast ^The Dura position does not have the sign of the tissue of connective tissue.On the tissue of implanting and below have inflammatory exudation, and have the inflammatory infiltration in the transition region of the dura mater of molding in advance.Subarachnoid space is detectable and tangible.Referring to Figure 15.

In eight weeks after operation, in described horse collagen thin slice group, the inflammatory process in new dura mater further fails.Have only little lymphocyte and mononuclear cell infiltration clump still to exist.Described subarachnoid space is clearly, and equally only has little inflammatory activity focus.Between the collagen fiber of the endogenous dura mater of height collagen and the new new dura mater that forms, show tangible seriality with EVG and the painted section of trichrome.This new film looks the thickness difference, and partly shows short texture.Referring to Figure 16.

At Tutoplast ^Inflammatory activity has also been eliminated at the Dura position well.There is not integration with adjacent dura mater at certain position, and, exists and the inflammatory infiltration of adjacent dura mater and the sign of adhesion in other positions.

After 16 weeks, in described horse collagen thin slice group, still can see the clump of lymphocyte and mononuclear cell nodositas inflammatory penetrant.Seriality between the actual dura mater of height collagen and the new dura mater does not change, and wherein great structural change do not occur with respect to the discovery in 8 weeks.Wherein there is some short texture in the collagen fiber that have different-thickness at certain position.

Tutoplast ^The Dura position has shown and the inflammatory variation and the adhesion of dura mater on every side equally; The intensity difference of these discoveries.

After 24 weeks, in described horse collagen thin slice group, except the further decline of cellular inflammation reaction, compare with the graft integration stage in 16 weeks, there is not relevant histology's difference.Referring to Figure 17.

Histology result

Provide in the table 1 below under transplantation site and dura mater and the tissue of the inflammatory reaction of epidural space, connective tissue and foreign body reaction degree quantitatively.

Show under 1-graft, the dura mater and the inspection of epidural space

		Horse collagen thin slice			TutoPlast Dura
										Under graft, the dura mater/epidural space			Under graft, the dura mater/epidural space
Time	Animal	Inflammation	Tissue	Foreign body reaction	Inflammation	Tissue	Foreign body reaction
The 2nd week	1	+++	--	+	++	--	++
									2	+++	--	++	++	--	+
	3	+++	--	+++	++	--	++
									4	+++	--	++	+++	--	+++
	5	+++	--	+++	++	--	++
The 4th week	6	++	u	+	++	--	+
									7	+	u	--	+	--	++
	8	+	u/K	--	+	--	+
									9	++	u	+	++	--	+
	10	+	u/K	+	++	--	+
The 8th week	11	+	K	+	++	--	+

		Horse collagen thin slice			TutoPlast Dura
										Under graft, the dura mater/epidural space			Under graft, the dura mater/epidural space
Time	Animal	Inflammation	Tissue	Foreign body reaction	Inflammation	Tissue	Foreign body reaction
									12	+	K	+	--	--	--
	13	++	K	u/+	++	--	+
									14	u/+	K	--	++	--	+
	15	+	K	u	--	--	+
The 16th week	16	--	K	--	+	--	--
									17	--	K	--	+	--	--
	18	--	K	--	--	--	--
									19	--	K	--	++	--	--
	20	--	K	--	+	--	--
The 24th week	21	--	K	--	+	--	--
									22	--	K	--	--	--	--
	23	--	K	--	--	--	--
									24	--	K	--	+	--	--

		Horse collagen thin slice			TutoPlast Dura
										Under graft, the dura mater/epidural space			Under graft, the dura mater/epidural space
Time	Animal	Inflammation	Tissue	Foreign body reaction	Inflammation	Tissue	Foreign body reaction
									25	--	K	--	--	--	--

Inflammation (inflammatory reaction):

--there is not the sign of visible inflammatory reaction

The inflammatory infiltration that u only limits to

The reaction of+mild inflammation

++ significantly inflammatory reaction

The inflammatory infiltration of +++serious

The tissue of graft:

--the tissue of connective tissue lacks or is slight

Isolated fibroblast in the+graft

The tissue (40%-70%) of the tissue of u limitation

Tissue＞70% of u/K tissue

The visible seriality of new dura mater of K and dura mater, the complete organization of graft (100%)

The reaction of foreign body and multinuclear giant cell:

--there is not foreign body reaction

U only has the foreign body reaction of limitation

+ moderate foreign body reaction

++ significant foreign body reaction

The foreign body reaction of +++widely

The histological examination result of the subarachnoid space of two transplantation sites is provided in the table 2 below.

The inspection of table 2-subarachnoid space (SAS)

		Horse collagen thin slice			TutoPlast Dura
										Subarachnoid space			Subarachnoid space
Time	Animal	Inflammation	Closed	Open	Inflammation	Closed	Open
The 2nd week	1	+	+	--	++	+	--
									2	++	+	--	++	+	--
	3	+++	+	--	++	+	+
									4	+++	+	--	+++	+	--
	5	++	+	--	++	+	+
The 4th week	6	--	--	+	+	--	+
									7	--	F	+	--	F	+
	8	--	F	+	--	F	+
									9	+	F	+	+	F	+
	10	--	--	+	+	F	--
														F
The 8th week	11	+	--	+	--	--	+
									12	+	u	+	--	--	+

		Horse collagen thin slice			TutoPlast Dura
										Subarachnoid space			Subarachnoid space
Time	Animal	Inflammation	Closed	Open	Inflammation	Closed	Open
									13	--	--	+	--	--	+
	14	--	u	+	--	--	+
									15	--	--	+	--	--	+
								The 16th week	16	--	F	--	--	F	--
	17	--	F	--	--	F	+
									18	--	F	--	--	F	--
	19	--	F	--	--	F	--
									20	--	F	--	--	F	--
								The 24th week	21	--	F	+	--	F	+
	22	--	F	+	--	F	+
									23	--	F	+	--	F	+
	24	--	F	+	--	F	+
									25	--	F	+	--	F	+

Inflammation:

--the NIP reaction

The reaction of+moderate inflammation

++ significantly inflammatory reaction

Serious inflammatory reaction

SAS (subarachnoid space) closure:

--dispersive inflammatory cell

+ by the SAS of Premeabilisation of cells closure

U is isolated inflammatory infiltration in SAS

Part fibrosis/fibrosis of pF/F SAS

SAS is open:

--SAS mainly is clearly, has isolated groups of cells

+ subarachnoid space is clear

Discuss

The processing of the assessment of operation method and two kinds of grafts

Described horse collagen thin slice is with the feature that is treated in the not in-problem operation.Hydration 5 minutes has once more produced the thick adamantine thin film of about 2mm in normal saline, and it can not lose its shape, can adhesive aggregation, and very easy excision forming.Be easy to this material be put into the dura mater defective with tweezers and tack hook.Because its activeness also was easy to correct described horse collagen thin slice in the lip-deep position of brain before being fixed on certain position.There is no need to sew up described thin film, because use the fibrin adhesive ﹠ can be with described graft fast and easily attached on the described dura mater border.Research in the past confirms also that equally fibrin adhesive ﹠ is the reliable sealer that is used for the dura mater closure.

Just the experimental fixedly stitching thread of using can be drawn back from described horse collagen thin slice with the dilatory of minimum, thereby show that fibrin adhesive ﹠ is the better choice of this graft.

When taking out described product subsequently, under some occasion, exist the too random fibrin adhesive ﹠ of using to cause sign with the covering bone adhesion of certain position already.These adhesions must be unclamped carefully with dissector, so that avoid spurring meninges and cortex.This problem can go up a spot of fibrin adhesive ﹠ by use or point and avoid.When taking out described product, clearly, using of fibrin adhesive ﹠ self produced firm dura mater closure, and avoided cerebrospinal fluid to ooze out.Just begin in this phenomenon 2 weeks after operation to occur, at this moment, do not have animal to develop subcutaneous CSF and leak or the cerebrospinal fluid fistula.

Through after short time of a few minutes rehydrated, Tutoplast ^The Dura product is easy to handle equally, but it is harder, and shows big fluctuation aspect bore.

Fibrin adhesive ﹠ produces the adhesion of appropriateness, so that with Tutoplast ^Dura remains on the initial position, but when reopening operative site, the connection between original dura mater and the graft is loosened.24 weeks still were this situations after operation, and this is because Tutoplast ^The result that Dura itself does not merge with dura mater from body.Therefore as if, the practice that this graft and single stitching thread are adhered to is better than the use fibrin adhesive ﹠, show because observe, and suitable integration can not occur under other occasions.At Tutoplast ^Adhesion seldom appears in the transition region between Dura and the cortex, and these adhesions use dissector to be easy to separately.

As if the technology that is used for described horse collagen thin slice is inserted between dura mater and the cortex be unaccommodated under some operation occasion.Specifically, relating to the big defective of brain essence in this way, for example, as if can not suitably fix in the operation in tumor chamber.

On the macroscopy level, all animals all show the reliable closure of dura mater defective, wherein do not have transplant rejection.In the little adhesion that has developed under the minority occasion between graft and the cortex construction, this may be owing at intra-operative arachnoid small damage is caused.Excessively the abuse fibrin adhesive ﹠ causes some animal to have adhesion that unclamp easily and bone imbrication in little zone.

From histology's angle, the intensive infiltration to described horse collagen thin slice of lymphocyte, macrophage and fibroblast appearred after implanting in 14 days.In graft, formed capillary tube subsequently.4 weeks only after operation, under arachnoidea and under the dura mater/inflammatory of following of epidural space just changes fully decline.Successive transition that occur between described horse collagen thin slice graft and dura mater on every side is visible at this moment equally because collagen fiber are newborn.

Thick not as original dura mater by the inductive this new dura mater of described horse collagen thin slice 24 weeks after operation, this may be because described dura mater thin slice only provides certain thickness from beginning.But this species diversity may be cancelled along with being created in subsequently of more collagen fiber.

Two weeks after operation, Tutoplast ^Dura shows the macroscopic encapsulation of described product in the thin layer connective tissue, described thin layer is thickening As time goes on.In addition, all animals all show suitable dura mater closure, do not have the CSF fistula, and a little adhesion occurs between graft and cortex or bone.

Although in the structure around the graft, have similar inflammatory reaction, the not sign of organizing after the operation, and the sign of the Premeabilisation of cells of rare graft and recovery.

Except lymphocyte and mononuclear cell inflammatory reaction that the part of expecting comprises, there is not the unusual or wound infection of animal development neurological.

The swelliong power of embodiment 2-horse collagen thin slice

The research that relates to the swelliong power of described horse collagen thin slice is carried out by the following method:

1) at first described horse collagen thin slice is cut into 1cm ²Square plate.

2) at the sample of these cutting blades of conventional sweep test under microscope, so that determine the gross morphology concordance and the thickness of described material, as the reference of expansion process.

3) then these cutting blades are put into plastic culture dish.

4) check fluid absorbency and swelliong power then in the following manner: use the normal saline solution Continuous Titration, use, wherein use to add a large amount of fluids gradually, from 10 μ l/cm with the Eppendorf micropipette ²Beginning is up to the highest 150 μ l/cm ²(referring to table 3).

Table 3

	Be administered to the fluidic amount of horse collagen thin slice
										Time	10μl/cm 2	20μl/cm 2	30μl/cm 2	40μl/cm 2	50μl/cm 2	75μl/cm 2	100μl/cm 2	125μl/cm 2	150μl/cm 2
1 hour	No bulking effect	Fluid immerses material fully, does not have supernatant after 1 hour	Fluid immerses material fully, does not have supernatant after 1 hour	Fluid immerses material fully, does not have supernatant after 1 hour	10 μ l supernatant	25 μ l supernatant	50 μ l supernatant	75 μ l supernatant	100 μ l supernatant
										2 hours	No bulking effect	Fluid immerses material fully, does not have supernatant after 2 hours	Fluid immerses material fully, does not have supernatant after 2 hours	Fluid immerses material fully, does not have supernatant after 2 hours	10 μ l supernatant	25 μ l supernatant	50 μ l supernatant	75 μ l supernatant	100 μ l supernatant
3 hours	No bulking effect	Fluid immerses material fully, does not have supernatant after 3 hours	Fluid immerses material fully, does not have supernatant after 3 hours	Fluid immerses material fully, does not have supernatant after 3 hours	10 μ l supernatant	25 μ l supernatant	50 μ l supernatant	75 μ l supernatant	100 μ l supernatant

The result:

As above measuring the Fluid Volume that is penetrated in the described horse collagen thin slice after 1,2 and 3 hour shown in the table.

A slice size is 1cm ²Horse collagen sheeting can absorb the brine fluids of 10 μ l amount fully, and do not have the obvious visible expansion of thickness.

The brine fluids of 20 μ l amount can enough 1cm ²Horse collagen thin slice absorb fully, and cause the increase slightly of this material thickness.

In whole series, only observed the increase of the minimum of horse collagen sheet thickness.According to estimates, the maximum increase of thickness only is about 2 times of initial volume, even after 3 hours.

After first hour, do not find the remarkable increase of thickness or absorption of fluids.

The increase of the length of the horse collagen thin slice of embodiment 3-hydration

1.0cm ²Seven dry horse collagen thin slices of size were placed in the isotonic sodium chloride hydration 1 hour.The average length that causes owing to the hydration of dry horse collagen thin slice increases to about 3.4%.

Table 4

The sheet numbering	Dry length (mm)	Hydration length (mm)
			1	17.5	18
2	17.7	18.3
			3	17	17.9
4	17.8	18.5
			5	18.1	18.7
6	18.6	19.5
			7	17.7	18.2
On average	17.8	18.4

The increase of the horse collagen flake weight of embodiment 4-hydration

With size is 1.0cm ²Seven exsiccant horse collagen thin slices be placed in the isotonic sodium chloride hydration 1 hour.The weight of the horse collagen thin slice of hydration is approximately five times of drying regime.

Table 5

The sheet numbering	Dry length (mg)	Hydration length (mg)
			1	8.4	40.2
2	7.6	37.9
			3	8	39.3
4	8.1	39.6
			5	8.6	41.9
6	8.7	46.8
			7	7.7	38.3
On average	8.2	40.6

The tensile strength and the elasticity/flexibility of embodiment 5-horse collagen thin slice

Measured the tensile strength of some dura mater products and horse collagen thin slice of the present invention.Sample is installed in the lower end of pipe, and water column increased continuously is 300cm to the maximum and improves tensile strength.Figure 18 provides the explanation of test chamber.

Described test chamber can be tested and appraised product owing to the point that the pressure that exceeds this product tensile strength damages, and measures the intensity of described dura mater succedaneum.Horse collagen thin slice (collagen content: 5.6mg/cm of the present invention ²) and collagen thin slice (collagen content: 4.0mg/cm ²) (IntegraNeuroSciences, Plainsboro NJ) compare with Duragen.

Result of the test shows, described horse collagen thin slice (collagen content: 5.6mg/cm ²) and collagen thin slice (collagen content: 4.0mg/cm ²) can bear the water column that is up to 300cm and do not damage.Referring to Figure 19 and 20.Comparatively speaking, the pressure that produces in healthy skull can not surpass the high water column of about 15cm; Under pathologic state, described pressure can be brought up to the highest about 50cm.

DuraGen has measured significantly lower tensile strength, breaks under the pressure of 200cm water column.Referring to Figure 21.Described test chamber can also be tested and appraised the elasticity that degree that described product stretches is come more described product under pressure.Therefore, the elasticity/flexibility of product is to determine by the protrusion of measurement products under described water column weight.

With described horse collagen thin slice (collagen content: 5.6mg/cm ²) and collagen thin slice (collagen content: 4.0mg/cm ²) elasticity/flexibility and DuraGen, collagen test sheets, Tutoplast fascia lata (Tutogen Medical GmbH, Neunkirchen am Brand, Germany) and Ethisorb Dura Patch (Ethicon GmbH ﹠amp; Co.KG, Nordrstedt. Germany) compare.Referring to Figure 22-25.

Compare described horse collagen thin slice (collagen content: 5.6mg/cm with other products ²) show as bigger tensile strength and the link coupled mixture of elasticity/flexibility.This makes it can bear the pressure that produces as the dura mater succedaneum on it, keeps pliability and elasticity simultaneously, thereby makes it be fit to the profile of brain and skull.On the contrary, Ethisorb and Tutoplast show high tensile strength in the water column test, but have much lower elasticity/flexibility.

The liquid-tight characteristic of embodiment 6-

The measurement of the liquid-tight characteristic of displacement dura mater product is to use the identical experiment of being discussed with embodiment 5 that mensuration is set.

In this experiment, the volume of the water of the development of relative water-column measurement water droplet and forfeiture.This result of experiment is shown in Figure 23-26.

In this experiment, described horse collagen thin slice (collagen content: 5.6mg/cm ²) to keep under the high water column of 300cm be liquid-tight surpassing.Described collagen thin slice (collagen content: 4.0mg/cm ²) forfeiture that under the high water column of 300cm, shows little water droplet.Owing to have loose structure, DuraGen is not fluid-tight, but even the forfeiture that under low water pressure, also shows tangible water.Similarly, the Tutoplast fascia lata is not fluid-tight equally, the forfeiture that shows water under low-pressure.

Embodiment 7-stability and tear strength

Only material enough stable, flexible and anti tear is suitable as the implant of particular procedure occasion under moist and dry environment, as is used for substituting dura mater.Therefore, measure the tear strength/limit tension of moist material, provide about structure, stability and remain on the valuable information of the probability of operative site.

With the tear strength of hydration sample test collagen surgery implant, so that simulation dominant condition in health.Described material was put into normal isotonic saline solution 5 minutes.

In order to measure tear strength/limit tension, with the collagen implant bar at Zwick Model 1120All-Purpose Testing Machine (Zwick GmbH ﹠amp; Co.KG, Ulm, Germany) in clamp.The collagen implant of testing is provided in table 6.

Table 6-collagen implant

Test strip	Reagent	Concentration	The source
				A	Collagen " compression " sponge	10.0mg/cm 2	Cattle corium
B	Collagen " foam " sponge	About 2.6mg/cm 2	The horse heel string
				C	Collagen sponge	2.8mg/cm 2	The horse heel string
D	The collagen thin slice	4.0mg/cm	The horse heel string
				E	Horse collagen thin slice	5.6mg/cm	The horse heel string

Machine control, data are obtained and test, and comprise that statistics estimates to be to use TestExpert software (Zwick GmbH ﹠amp; Co.KG, Ulm, Germany) carry out.Test sponge sample A, B and C it seems quite fragile.Sample A, B and C are cut into the bar that width is the 4cm of 1.4cm.Calculate the test result of the 1.0cm width bar of all samples in proportion.The limit tension value of test strip is that unit is measured with newton/cm-bar.In table 7 and Figure 27, provide test result.

Table 7-tear strength/limit tension

Test strip	Tear strength/limit tension (/the cm-bar)	Standard deviation
			A	0.46	0.19
B	0.45	0.11
			C	1.64	0.69
D	3.21	0.69
			E	4.09	0.24

Conclusion

The production method of the uniqueness of described horse collagen thin slice has improved its tear strength/limit tension.

Compare with collagen sponge, the collagen thin slice shows obviously higher tear strength/limit tension.

The tear strength of collagen thin slice improves along with the raising of every square centimeter collagen content that (for example, collagen content is 4.0mg/cm ²: the 3.21N/cm-bar; Collagen content is 5.6mg/cm ²: the 4.09N/cm-bar).

In sum, some purposes of the present invention have been realized as can be seen.

Owing under the prerequisite that does not deviate from the scope of the invention, can carry out multiple change, so wish that all items that comprised in above-mentioned description all are understood as that illustrative rather than restriction character to above-mentioned composition and method.

" comprise (comprise) " or " comprising (comprises) " or " comprising (comprising) " about employed word in whole description (claims below comprising), the applicant points out, unless other needs are arranged in context, these words are with basic and clearly understand to use, be that they are interpreted into and comprise character, rather than exclusive character, and the applicant wishes the explanations by this way in understanding whole description of above-mentioned each word.

Claims (27)

1. Horse collagen thin slice production be used for by described dura mater tissue is contacted with described thin slice repair and the mammal that regenerates in purposes in the medicine of dura mater tissue, wherein, described horse collagen thin slice comprises the bio-matrix that the non-natural that is not the horse collagen fibril by chemical reagent or crosslinking with radiation exists, described bio-matrix is an atresia substantially, and become to be grouped into by acellular basically, described composition comprises connective tissue protein.
2. 2. purposes as claimed in claim 1, wherein, described horse collagen thin slice comprises the multilamellar collagen fibril.
3. 3. as the purposes of claim 1 or 2, wherein, described horse collagen fibril comes from tendon.
4. 4. purposes as claimed in claim 3, wherein, described tendon is a heel string.
5. 5. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice is can be resorbent.
6. 6. as any one purposes in the above-mentioned claim, wherein, described dura mater tissue needs to repair and regeneration because of congenital situation, birth defect, disease, damage or operation process.
7. 7. purposes as claimed in claim 6, wherein, described operation process is a tumor resection.
8. 8. as any one purposes in the above-mentioned claim, wherein, described dura mater tissue is positioned at skull.
9. 9. as any one purposes among the above-mentioned claim 1-7, wherein, described dura mater tissue is positioned at spinal column.
10. 10. as any one purposes in the above-mentioned claim, wherein, the thickness of described horse collagen thin slice under dried forms is 0.01mm-3.0mm, for example 0.02mm-2.0mm, 0.03mm-1.5mm or 0.05mm-1.0mm.
11. 11. as any one purposes among the above-mentioned claim 1-9, wherein, the thickness of described horse collagen thin slice under its dried forms is 1.0mm or thinner.
12. 12. as any one purposes in the above-mentioned claim, wherein, described contact procedure comprises utilizes fibrin sealer, tissue glue and/or surgical sutures, and/or utilize the pressure fitted technology or utilize horse collagen thin slice and the dura mater tissue between natural adhesion with described horse collagen thin slice attached to the dura mater tissue.
13. 13. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice is liquid-tight substantially.
14. 14. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice hydration 5 seconds to 10 minute for example before described contact procedure, preferably before described contact procedure in normal saline hydration 1-6 minute.
15. 15. as any one purposes among the above-mentioned claim 1-13, wherein, described horse collagen thin slice did not carry out hydration before described contact procedure.
16. 16. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice is when complete hydration, weight reaches maximum 15 times of its dry weight, preferably only reaches maximum 5 times of maximum 10-of its dry weight.
17. 17. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice its weight when drying is 1mg/cm ²-50mg/cm ², for example, 2.5mg/cm ²-10mg/cm ²
18. 18. as any one purposes among the above-mentioned claim 1-16, wherein, the surface area of surface area ratio its dried forms of described horse collagen thin slice when complete hydration is big-5%-20%, preferred big-5%-10% or-5%-5%, for example than its dried forms larger about 4%.
19. 19. purposes as claimed in claim 1, wherein, the thickness of described horse collagen thin slice approximately is about 2 times or 3 times of thickness of its dried forms when complete hydration.
20. 20. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice can be after carrying out cell tissue attached to nervous tissue or cerebral tissue with meningocyte on.
21. 21. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice with meningocyte generation cell tissue after can be attached to skull or spinal tissues on.
22. 22. as any one purposes in the above-mentioned claim, wherein, described mammal is selected from: people, horse, sheep, monkey and laboratory animal.
23. 23. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice also comprises and is selected from following one group excipient: antiseptic, somatomedin, the pliability that helps horse collagen thin slice and elastic additive, and their combination.
24. 24. as any one purposes in the above-mentioned claim, wherein, described horse collagen thin slice does not comprise virus or Protein virus.
25. 25. as any one purposes in the above-mentioned claim, wherein, described collagen fibril is made up of type i collagen basically.
26. 26. as any one purposes, the wherein cell tissue of meningocyte about six all complete organizations after operation in the above-mentioned claim.
27. 27. as any one purposes in the above-mentioned claim, wherein, the limit tension of described horse collagen thin slice is 0.5 newton/cm-bar-30 newton/cm-bar, for example, and 1 newton/cm-bar-6 newton/cm-bar.

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