CN105288736B - Acellular dermal matrix cornea and the preparation method of ocular tissue renovation material - Google Patents
Acellular dermal matrix cornea and the preparation method of ocular tissue renovation material Download PDFInfo
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- CN105288736B CN105288736B CN201510827017.9A CN201510827017A CN105288736B CN 105288736 B CN105288736 B CN 105288736B CN 201510827017 A CN201510827017 A CN 201510827017A CN 105288736 B CN105288736 B CN 105288736B
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Abstract
The invention discloses a kind of acellular dermal matrix cornea and the preparation method of ocular tissue renovation material, it comprises the following steps: step A: choose the dermal tissue at certain position of mammalian body or human body；Step B: by contrasting the structure of described dermal tissue and cornea tissue, determine the dermal tissue β Rotating fields with close and adjacent 5 layers of described cornea tissue structure in described dermal tissue structure；Step C: remove other layers in addition to dermal tissue β Rotating fields in described dermal tissue, it is thus achieved that dermal tissue β layer primary raw materials.The preparation method of the present invention has been widened and has been prepared cornea and the raw material sources of ocular tissue renovation material, reducing production cornea or the cost of ocular tissue renovation material, the acellular dermal matrix cornea of preparation can be widely applied to infection, wound or lesion tissue and causes the cornea reproducibility reparation of corneal injury.
Description
Technical field
The present invention relates to a kind of acellular dermal matrix cornea and the preparation method of ocular tissue renovation material.
Background technology
At present, cornea pathological changes is frequently-occurring disease, commonly encountered diseases, especially wound, infection local organization pathological changes, crystal pathological changes meeting
Causing cornea edge to change, the final transparency changing cornea tissue causes blind.It is blind that keratopathy causes, the most only
One treatment means implemented is corneal allograft, deficient owing to contributing material source, the patient of a large amount of corneal transplantation cases because of
Wait that corneal transplantation is in blind state.Show according to ophthalmology report on Epidemiological in 2011: China is because of keratopathy
Cause blind patient to be about 7/100000ths. one annual, i.e. annual increase about 100,000 examples.China can be used for moving every year at present
The allogeneic cornea material planted less than 10,000, the number of patients of accumulative waiting angle film healing treatment about 2,000,000 examples, if
Count because part keratopathy or Perforation pathological changes need this number of patient of corneal material or ocular tissue renovation material
Amount is up to up to ten million.
At present, the most all carrying out the research of substantial amounts of cornea substitute products, cornea substitute products material is concentrated mainly on
Allosome amniotic membrane, marine organisms collagen protein and heterogenic cornea material aspect, above-mentioned material due to material self-characteristic restriction all
It is extremely difficult to the permanent or effect of recovering lost eyesight property.
As can be seen here, the demand of domestic and international corneal succedaneum and ocular tissue renovation material is big, existing product
Not there is the permanent or effect of recovering lost eyesight property, it would be highly desirable to the cornea repair material of the bioengineered tissue that exploitation is new further.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of acellular dermal matrix cornea and ocular tissue renovation material
Preparation method, this preparation method its widened and prepared cornea and the raw material sources of ocular tissue renovation material, reduce life
Producing cornea or the cost of ocular tissue renovation material, the acellular dermal matrix cornea of preparation can be widely applied to infection, wound
Or lesion tissue causes the cornea reproducibility reparation of corneal injury.
For solving the above-mentioned technical problem present invention a kind of acellular dermal matrix cornea and the system of ocular tissue renovation material
Preparation Method, it comprises the following steps:
Step A: choose the dermal tissue of mammalian body or human body；
Step B: by contrasting the structure of described dermal tissue and cornea tissue, determine in described dermal tissue structure with institute
State the dermal tissue β Rotating fields of close and adjacent 5 layers of cornea tissue structure；
Step C: remove other layers in addition to dermal tissue β Rotating fields in described dermal tissue, it is thus achieved that dermal tissue β layer
Primary raw materials；
Step D: described dermal tissue β layer primary raw materials is carried out de-cell and processes, it is thus achieved that at the beginning of acellular dermal tissue β layer
Level raw material；
Step E: by the acellular dermal tissue β layer primary raw materials that process through step D under aseptic, constant temperature, Yu Ping
Weighing apparatus liquid mixes and shakes certain time, restore collagen protein, the biology of active factors in described acellular dermal tissue β layer primary raw materials
Performance；
Step F: by the acellular dermal tissue β layer primary raw materials after processing in step E, processes in balance liquid, logical
Cross the permeability changing albuminous membranae and the mode supplementing balance ion, close the potential point of dermal tissue β layer primary raw materials,
To acellular dermal matrix cornea and ocular tissue renovation material.
In one embodiment of the present of invention, in described step F, come described in leveling de-by combined ionic or single ionic
The potential difference of the dermal tissue β layer primary raw materials after cell, reaches the dermal tissue β layer primary raw materials after closing described de-cell
The purpose of the potential point of middle protein.
In one embodiment of the present of invention, in described step C, remove in dermal tissue structure except dermal tissue β Rotating fields
During other layers in addition, retain nerve growth factor and nestin-entactin, be beneficial to set up the non-vascular battalion of cell
The pattern of supporting.
In one embodiment of the present of invention, in described step D, remove described dermal tissue β layer primary raw materials has and exempt from
The epidermis cell of epidemic focus and other adult cell, retain collagen protein and the elastic force fibre with natural tissues space framework
Dimension, and maintain the de-cell pre-structure of dermal tissue β layer primary raw materials.
In one embodiment of the present of invention, in described step C, utilize fixative to described dermal tissue β layer primary raw materials
It is fixed.
In one embodiment of the present of invention, in described step E, in described constant temperature, the temperature range of choice is that 16-37 takes the photograph
Family name's degree.
In one embodiment of the present of invention, in described step E, in balance liquid, the mixed time shaken is 4-24 hour.
In one embodiment of the present of invention, prepared acellular dermal matrix cornea or ocular tissue renovation material are direct
Transplanting for human or animal.
The preparation method of acellular dermal matrix cornea of the present invention and ocular tissue renovation material has the advantages that
The present invention provides in the preparation method of acellular dermal matrix cornea and ocular tissue renovation material, choose animal or
The corium of person's human body, as raw material sources, has expanded the available sources of raw material, reduces the cost preparing cornea substitution material, and
The preparation method of the present invention is simple, and low cost of manufacture, this acellular dermal matrix cornea and ocular tissue renovation material can be extensive
It is applied in the cornea reproducibility reparation that infection, wound or lesion tissue cause corneal injury.
Below by drawings and Examples, technical scheme is described in further detail.
Accompanying drawing explanation
Fig. 1 is that the acellular dermal matrix corneal material implantation experimental rabbit of the present invention changes the aobvious of growing state over time
Micro mirror photo figure；
Fig. 2 is that the acellular dermal matrix corneal material prepared of the present invention implants the Histological section of 12 hours after animal body
Photo figure；
Fig. 3 is that the acellular dermal matrix corneal material prepared of the present invention implants the Histological section of 72 hours after animal body
Photo figure；
Fig. 4 is the acellular dermal matrix corneal material prepared of present invention Histological section when implanting after animal body 7 days
Photo figure；
Fig. 5 is the acellular dermal matrix corneal material prepared of present invention photo figure when implanting animal corneal position 6 weeks；
Fig. 6 is the acellular dermal matrix corneal material prepared of present invention photo figure when implanting animal corneal position 24 weeks.
Detailed description of the invention
Acellular dermal matrix cornea of the present invention and the preparation method of ocular tissue renovation material, it comprises the following steps:
Step A: choose the dermal tissue of mammalian body or people.
Mammalian body include pig, cattle, etc., it is also preferred that the left the dermal tissue chosen on human body, mammal different parts
The function of corium there are differences, and stromatin contained by different parts corium and the type of the various factor, quantity are different.According to not
With the composition structure of position corium, function and the difference of various factor content, select the corium of corresponding site targetedly.
Step B: by the structure of dermal tissue described in relative analysis Yu cornea tissue, determines and chooses in dermal tissue structure
Dermal tissue β Rotating fields with close and adjacent 5 layers of described cornea tissue structure.
Cornea tissue structure can be divided into 5 layers substantially, respectively epithelial layer (epitthelial layer), bowman's lamina
(Bowman's membrane), hypothallus (Stroma), descemet's membrane (Descemet's membrane) and endodermis
(Endothelium layer).The each layer of cornea tissue be mainly composed of functioning cell and I, II, III, VI collagen type, glue
The layering of former albumen arranges in an orderly manner, and wherein, VI collagen type exists with network structure, and the ordered arrangement of corneal substrate also rises
Important function.Unified and queueing discipline the NTx albumen of diameter is the key factor maintaining corneal transparency.
The present invention is compared, to collagen protein class by ultramicroscope corneal structure and substantial amounts of skin corium structure
The qualitative comparison of type, the orientation order of collagen protein is compared, and determines and cornea group in 250 layers of dermal tissue structure
Knitting 5 Rotating fields that structure is the most close and adjacent, the present invention is by this adjacent 5 Rotating fields named dermal tissue β Rotating fields.
Step C: remove other layers in addition to dermal tissue β Rotating fields in dermal tissue structure, it is thus achieved that dermal tissue β layer
Primary raw materials.
Healthy corium is being treated in journey, dermal tissue β layer primary raw materials is being fixed by fixative, right
The somatomedin etc. of the collagen protein and attachment that constitute frame structure is protected, except corium group in removing dermal tissue structure
During knitting other layers beyond β Rotating fields, retain nerve growth factor (NGF nerve growth factor) and nest egg
In vain-entactin (N-E nidogen-entactin), acellular dermal matrix cornea and the ocular tissue renovation material of preparation are planted
After entering animal body, can the growth of accelerator nerve fiber, the edge of cornea organizational structure that fast quick-recovery is destroyed, be conducive to setting up thin
The non-vascular Model of Nutrition of born of the same parents.
Step D: the dermal tissue β layer primary raw materials in described dermal tissue structure is carried out de-cell and processes.
Dermal tissue β layer primary raw materials carries out de-cell process, use tissue engineering to separate with biochemical method,
The epidermis cell destroy, removed in dermal tissue and other adult cell etc. have immunogenic material, and reservation has natural
The materials such as the collagen protein of tissue space framework and elastic fibers, and maintain knot before the de-cell of dermal tissue β layer primary raw materials
Structure.
Concrete, liquid is prepared, wherein in the basis that the present invention utilizes in Patent No. CN1130971C in de-cell technology
Adding certain enzyme and chemical reagent is prepared as de-cell and extracellular matrix fixes preparation, fixative is primary with dermal tissue β layer
Raw material is mixed to be postponed and carries out that constant temperature is mixed shakes operation, dermal tissue β layer primary raw materials carries out de-cell and processes, obtain de-cell and process
After dermal tissue β layer primary raw materials.
Step E: by the dermal tissue β layer primary raw materials that obtain in step D under aseptic, constant temperature, this constant temperature
Temperature range be 16-37 degree Celsius, be placed in balance reducing solution and carry out mixed shaking operation 4-24 hour, restore after de-cell processes
The collagen protein of dermal tissue β layer primary raw materials, the biological property of active factors.
In step E, it is also preferred that the left be 25 degrees Celsius in temperature, in balance liquid, mixed shaking operates 20 hours.
As disposable embodiment, in this step, under 32 degrees Celsius of constant temperatures, in balance liquid, mix and shake behaviour
Make 24 hours.
As disposable embodiment, in this step, under 16 degrees celsius, carry out mixing in balance liquid and shake operation
4 hours.
Step F: process processing the dermal tissue β layer primary raw materials obtained in step E in balance liquid, with by changing
Albuminous membranae permeability and the mode of supplementary balance ion, close the potential point of dermal tissue β layer primary raw materials so that corium group
Knitting all potential points of albumen in β layer primary raw materials is all 0, removes the collagen protein signal of telecommunication pair in dermal tissue β layer primary raw materials
The impact of cell transformation, obtains can be applicable to the acellular dermal matrix cornea that human cornea is repaired so that acellular dermal base
After matter corneal material implants human body, after host cell enters primary raw materials frame gap, its differentiation direction is not by acellular dermal
The interference of intrastromal corneal material itself.
The present invention passes through combined ionic leveling potential difference, reaches to close dermal tissue β layer primary raw materials protein potential point
Purpose, it is also preferred that the left the present invention uses single ionic leveling potential difference, thus close the potential point of protein in primary raw materials,
Enter process further, finally give the acellular dermal matrix corneal material of the present invention.
The precursor of the reparation of cornea tissue is mainly derived from edge of cornea stem cell, and edge of cornea stem cell passes through water
Flat centripetal movement and the mode be combineding with each other moved vertically upward enter cornea and Ocular surface healing material gap.Through TAC
(transit amplif-ying cells transit amplifying cells), PMC (post-mitotic cells postmitotic cells)
It is eventually converted into TDC (terminally differentiated cells terminal cell).Corneal limbal cells is to functional
During cell transformation, closed on cell, local potential, fibronectin (FN) content and transforming growth factor-β
Multifactorial impacts such as (Transforming growth factor-beta TGF-β), the present invention passes through above-mentioned manufacturing step
Create the correlation factor in felicity condition protection dermal tissue β Rotating fields and electro physiology environment achieves edge of cornea stem cell
Precisely repair.
Additionally, the present invention can prepare non-cornea tissue through above-mentioned de-cell and collagen protein correlation factor resist technology
Acellular dermal matrix corneal material and ocular tissue renovation material, by select different parts leather material, mutually deserved
To acellular dermal matrix corneal material or ocular tissue renovation material.
Acellular dermal matrix cornea prepared by the present invention or ocular tissue renovation material, be subject to for mammalian tissues
During damage (include pathologic damage and think that actively destruction is damaged), utilize tissue engineering patented technology induction host precursor thin
Born of the same parents enter framework, and after repair materials implants human cornea position, in tissue liquid and blood, self stem cell and precursor are thin
Born of the same parents stick in entering material frame gap be detained, under periocular tissues electricity irritation and the factor act on jointly, stem cell and precursor are thin
Born of the same parents' multiple fission also converts to keratocyte, secretes new extracellular matrix, strengthens reparation, plasticity cornea tissue, in damage office
The cell in portion changes, chemotactic factor, basic fibroblast growth factor (Fibroblast growth factor
FGF), NTx protein promoter etc. have inducing cell to move to damage location, break up and value-added function.Corneal epithelium
The nutrition of cell stops blood vessel to spread growth to cornea due to the special construction of edge of cornea tissue, therefore corneal epithelial cell master
Corneal nerve to be relied on provides metabolic nutrition and supporting function, is converted into keratocyte under electricity physiological signal stimulates, and gradually
Set up the non-vascular Model of Nutrition utilizing aqueous humor to provide cytotrophy.
Acellular dermal matrix cornea repair material prepared by embodiment 1 present invention and ocular tissue renovation material bio-compatible
Property experiment.
Please refer to shown in Fig. 1, after acellular dermal matrix cornea prepared by the present invention implants experimental rabbit, experimental rabbit exists
The most normally survive during experiment, without severe complication.Do not find serious anterior chamber's inflammation.Acellular dermal matrix cornea implants it
Just there is edema phenomenon, eliminate after 12 weeks.Within after implantation 24 weeks, become corneal transparency.Early postoperation part disease eye occurs slight new
Vascularization phenomenon, vanished from sight after 12 weeks.
H&E dyes display, and the small circle cell in postoperative 1st week experimental rabbit cornea gathers at implantation near two ends, and the 12nd
Week and within the 24th week, have no.Having grown at implantation to the 24th week some fusiform cells, the growth of keratocyte, can be observed simultaneously
Implant border some part to have obscured, the collagen of fuzzy place's acellular dermal matrix material (acellular ADM) like the most with
The collagen of main body cornea is integrated.Collagen component is only dyeed by Picro-Sirius red, the acellular dermal matrix material of postoperative implantation
In ADM there is notable change in collagen structure.Before implanting, the collagen fiber of acellular dermal matrix materials A DM are relatively thick, complete, staggered
Arrangement, major part takes on a red color or bright yellow, and the collagen fiber of normal cornea are relatively thin, in yellow green or redness, regularly arranged.Art
Within latter 1 week, ADM is high-visible with the boundary of cornea, and color is greener, intersection has obvious black gap, the most then
Invisible.By postoperative 12nd week, the collagen fiber of implanted acellular dermal matrix materials A DM were thinned out, more in green, still
Dark gap be can be observed, but gap substantially diminishes, border starts to obscure.Had a common boundary fuzzyyer by the 24th week, can not see deep
Color gap, under micropolariscope observe acellular dermal matrix materials A DM color substantially become with around cornea more phase
Seemingly.
Please refer to shown in Fig. 2, acellular dermal matrix cornea prepared by the present invention is implanted after animal body 12 hours
Histological section, initially entering in material is inflammatory cell and fibrocyte, and in material, cell quantity and density ratio are relatively low.
Please refer to shown in Fig. 3, the present invention prepares acellular dermal matrix cornea and implants the group of 72 hours after animal body
Knitting section, cell density and quantity substantially increase, and disappeared on the cell type entered in addition to inflammatory cell fiber
Cell, but it appeared that grow more inmature precursor or stem cell.
Please refer to shown in Fig. 4, acellular dermal matrix cornea prepared by the present invention implants tissue when after animal body 7 days
Learn section, it can be seen that in material, cell density is already close to normal structure, based on mesenchymal cell or stem cell, and can
To see each phase form of cell division hypertrophy, the most can find and have the vascular tissue of complete structure.
Acellular dermal matrix cornea prepared by the histology experiment explanation present invention of above early stage implants animal eye cornea
Behind position, cell can enter material gap, and can multiple fission and be converted into functioning cell in the material.
Acellular dermal matrix cornea prepared by embodiment 2 present invention implants animal body reparative experiment
Please refer to shown in Fig. 5, acellular dermal matrix cornea prepared by the present invention implants animal corneal position 6 weeks
Time, embedded material has become smooth, becomes translucent.
Please refer to shown in Fig. 6, acellular dermal matrix cornea prepared by the present invention implants animal corneal position 10 weeks
Time, embedded material substantially transparent, animal can regard thing.
Acellular dermal matrix cornea prepared by the present invention or ocular tissue renovation material, through zoopery and histology
Experiment shows, acellular dermal matrix cornea and periocular tissues because the characteristic of self natural stereochemical structure was when the 24th week, its
Organizational structure, collagen-type, local strength and elasticity are completely consistent with host cornea tissue, the acellular dermal of the present invention
Intrastromal corneal or ocular tissue renovation material, fully achieve the purpose replacing existing cornea product, expanded raw material sources,
Its preparation method is simple, reduces manufacturing cost, and the acellular dermal matrix cornea substitution material of the present invention is by leading keratopathy
Change, the clinical treatment of corneal restoration.
The above, for presently preferred embodiments of the present invention, not impose any restrictions the present invention, those skilled in the art's profit
Make a little simple modification, equivalent variations or modification with the technology contents of the disclosure above, all fall within protection scope of the present invention.
Claims (7)
1. an acellular dermal matrix cornea and the preparation method of ocular tissue renovation material, it is characterised in that include following step
Rapid:
Step A: choose the dermal tissue of mammalian body or human body；
Step B: by contrasting the structure of described dermal tissue and cornea tissue, determine in described dermal tissue structure with described angle
The dermal tissue β Rotating fields of close and adjacent 5 layers of membrane tissue structure；
Step C: remove other layers in addition to dermal tissue β Rotating fields in described dermal tissue, it is thus achieved that dermal tissue β layer is primary
Raw material；
Step D: described dermal tissue β layer primary raw materials is carried out de-cell and processes, it is thus achieved that acellular dermal tissue β layer primary is former
Material；
Step E: by the acellular dermal tissue β layer primary raw materials that process through step D under aseptic, constant temperature, in balance liquid
In mixed shake certain time, restore collagen protein in described acellular dermal tissue β layer primary raw materials, active factors biological
Energy；
Step F: by the acellular dermal tissue β layer primary raw materials after processing in step E, processes, by changing in balance liquid
Become the permeability of albuminous membranae and supplement the mode of balance ion, closing the potential point of dermal tissue β layer primary raw materials, taken off
Acellular dermal matrix cornea and ocular tissue renovation material；
Wherein, in step F, carry out the dermal tissue β layer after de-cell described in leveling by combined ionic or single ionic primary
The potential difference of raw material, thus close the potential point of protein in the dermal tissue β layer primary raw materials after described de-cell so that true
In skin tissue β layer primary raw materials, all potential points of albumen are all 0.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step F, carry out the dermal tissue β layer primary after de-cell described in leveling by combined ionic or single ionic former
The potential difference of material, reaches the purpose of the potential point of protein in the dermal tissue β layer primary raw materials after closing described de-cell.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step C, during removing in dermal tissue structure other layers except dermal tissue β Rotating fields in addition to, reservation
Nerve growth factor and nestin-entactin, be beneficial to set up the non-vascular Model of Nutrition of cell.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step D, remove and described dermal tissue β layer primary raw materials has immunogenic epidermis cell and other one-tenth
Somatic cell, retains collagen protein and the elastic fibers with natural tissues space framework, and maintains dermal tissue β layer primary raw materials
De-cell pre-structure.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step C, utilize fixative that described dermal tissue β layer primary raw materials is fixed.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step E, in described constant temperature, the temperature range of choice is 16 ~ 37 degrees Celsius.
Acellular dermal matrix cornea the most according to claim 1 and the preparation method of ocular tissue renovation material, it is special
Levy and be,
In described step E, in balance liquid, the mixed time shaken is 4 ~ 24 hours.
Priority Applications (1)
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