CN103127550A - Skeletal muscle whole organ acellular matrix, its preparation method and its derived medical products - Google Patents
Skeletal muscle whole organ acellular matrix, its preparation method and its derived medical products Download PDFInfo
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Abstract
The invention discloses a skeletal muscle whole organ acellular matrix, its derived medical products, such as particles, fluidized compositions, gels and active peptides, and preparation and use methods of the matrix and the medical products. The skeletal muscle whole organ acellular matrix completely reserves the skeletal muscle three-dimensional parallelly-arrayed skeletal muscle basement membrane ultra-structure, the vascular matrix network and muscle-tendon joints, reserves large amounts of bioactive components comprising growth factors, hyaluronic acid, glycosaminoglycan, laminins and the like, is highly affinity to muscle-derived stem cells, has the advantages of certain mechanical strength and toughness, thorough decellularization, no obvious immunological rejection, and no ethical or moral issues, and is a skeletal muscle regeneration scaffold and platform having the most advantages reported so far. The prepared skeletal muscle whole organ acellular matrix and its derived medical products can be used for the in-vitro and in-vivo construction and regeneration of the skeletal muscles, are suitable for restoring the skeletal muscle coloboma of various positions in various ranges, and are expected to solve the large-range skeletal muscle coloboma restoration and muscle regeneration problems.
Description
Technical field
The invention describes the full organ acellular matrix of skeletal muscle ((Whole organ acellular matrix, WOACM)) and the medical product that derives such as the preparation method and its usage of microgranule, fluidisation compositions, gel and bioactive peptide etc.Relate to the field and comprise that organizational project and regenerative medicine, surgery, biomaterial learn.
Background technology
Up to the present the treatment that skeletal muscle is damaged, the particularly damaged reconstruction of muscle on a large scale are still a clinical difficult problem.Skeletal muscle accounts for the 40-50% of human body weight.The Musculoskeletal wound is also one of modal wound of human body, and the war wound of the athletic injury of 30-55%, 50-70% all relates to skeletal muscle according to statistics.Slight Skeletal muscle injury, as various contusion or pull, skeletal muscle tissue can rely on sarcoplast (satellite cell) regenerated muscle fibers to realize self-regeneration.Yet damaged amount surpasses that 20% skeletal muscle wound is difficult to the practical function reparation and with cicatrization, the atrophy of distal end muscle denervation, dysfunction.For these patients, at present clinical standard care is from the body reparation of drawing materials, comprise that various musculo cutaneous flaps transplant, though can partly recover the damage zone function, have available muscle derived limited, tissue anatomical structure and biomechanics changes, for deficiencies such as district's damages; Do not meet the developing direction of surgery Wicresoft with " repair in trauma wound " yet.In a word, up to the present the treatment that skeletal muscle is damaged, the particularly damaged reconstruction of muscle on a large scale are still a clinical difficult problem.Regenerate by induced tissue and realize that the reconstruction that skeletal muscle is damaged meets the medical science needs on a large scale, this technology has broad prospects.
Extracellular matrix has so far best body Endoskeleton flesh regeneration induction effect, but still has the improvement needs.Skeletal muscle is the simple function organ, realizes its functional regeneration than the internal organs such as liver, lung are easier by organizational project and regeneration medicine technology in theory.In fact, the progress of organizational project skeleton flesh has lagged behind the fields such as liver and lung at present, and reason is that skeletal muscle has complicated ultrastructure, comprises three-dimensional skeletal muscle vessel of basilar membrane structure that be arranged in parallel, periodic, muscle-tendon joints etc., these structures are extremely difficult artificial imitation all.To possess the prerequisite of function be to innervate and enough blood vessel networks provide nutrition and oxygen to divide (myocyte be substance metabolism is very vigorous, high oxygen consumption cell) to skeletal muscle in addition, and present various external structure environment, comprise bioreactor, all be difficult to provide the required oxygen of Skeletal Muscle Cell growth promoter to divide and nutritional support.Have in addition research to find, the skeletal myoblast (Myoblast) that enzymic digestion obtains can be lost the myofibrillar ability of regeneration skeleton in separation and amplification procedure.Directly Muscle-derived Stem Cells being inoculated into damaged part does not have the stem cell survival of inoculation to reach 1% report so far.Above-mentioned difficulties is ordered about people's research could induce skeletal muscle tissue regeneration in vivo, accelerates skeletal muscle regeneration, reduces cicatrization as only providing biochemical signals to regulate local microenvironment.This respect foreign scholar has obtained the breakthrough that attracts people's attention, namely use acellular matrix (Acellular matrix, ACM) timbering material, as trees-Osima jacoti, Osima excavata (Small intestinal submucosa, SIS) substrate, bladder substrate (Urinary bladder matrix, UBM), realize in vivo skeletal muscle function regeneration.The composition of these ACM is extracellular matrix (Extracellular matrix, ECM), mainly formed by I type and III type collagen fiber, contain a small amount of V-type and VI type collagen fiber, certain porosity is arranged, remainder is mainly glycoprotein, Fibronectin, glycosaminoglycan (chondroitin sulfate, hyaluronic acid, heparin etc.), the growth and proliferation of cell that is easy to cell adhesion and can promotes to adhere to; Contain cytokine profiles, comprise fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-β and VEGF (VEGF) etc.; Also have good degradability and degradation rate controllability, without antigenic action.It is " endogenous tissue regeneration " that ECM repairs the damaged mechanism of skeletal muscle: after implanting, ECM can realize quick revascularization, biological activity signal protein wherein and somatomedin attract the stem cell migration to enter timbering material, and induced dry-cell is differentiated to form location specific parenchyma cell such as skeletal myoblast, blood vessel precursor; Along with the degraded of the ECM material that continues and the lasting newborn apposition of host cell, functional skeletal muscle tissue begins to form, and reverts to its source structure, functional and physiological state.Laboratory animal skeletal muscle defective region uses ECM to fill and repairs, although do not inoculate stem cell, also can bear functional skeletal muscle again.Valentin etc. use SIS to repair the rat abdominal-wall defect, distinguish in reparation after 6 months and see the functional skeletal muscle tissue of island shape, and the contractility of its generation can reach 80% of normal abdominal muscle.Equally, Turner etc. observe the skeletal muscle new life that the SIS reparation is distinguished has blood to supply well, innervate, have contractile function equally after 6 months in the large fraction muscle of dog 8 * 4cm lacks model, almost distinguish the differentiation of a surrounding normal muscular tissue difficult problem with reparation.Certainly, the pre-vaccination stem cell can further be accelerated tissue regeneration speed; Conconi etc. find again to implant reparation rat abdominal-wall defect after the external inoculation of rat abdominal muscle ECM sarcoplast, only can form in 9 days have labyrinth, the skeletal muscle tissue of collapsible, vascularization.Above-mentioned result of study has been converted into clinical practice abroad, namely implants the ECM material and promotes to suffer the patient of medium range skeletal muscle loss to realize that muscle function rebuilds, and improves physical disabilities.Existing report all comes from US military.The 1st routine clinical practice is that employing UBM is that the soldier that a right lower extremity gastrocnemius part of returning from Afghan battlefield is blown up has realized anathrepsis, and the skeletal muscle tension of injury returns to 70% of offside.The newspaper of China " Reference News " has also been reprinted this achievement (the 19120th phase, on June 21st, 2011).The 2nd example is the soldier of a right thigh root portion skeletal muscle disappearance, has 3 years apart from he is injured when he accepts multilamellar SIS implanted treatment.In these 3 years, his injured district is the cicatrix reparation, though through rehabilitation for many years, he still has obvious right knee joint to stretch difficulty always.Accept SIS and implant rear April, his this symptom is obviously improved, and injured district muscular strength has improved 30%.But it should be noted that, still there are some limitation at present in this method, comprise 1. complex operation, in above-mentioned research, ECM material used is UBM and SIS, although convenient sources, but because less than 100 μ m, reaching the volume of damaged skeletal muscle, the average thickness of UBM and SIS needs the material of stacked hundreds of layers; 2. the skeletal muscle of regenerating not is the skeletal muscle that truly has whole contractile functions: the skeletal muscle substance metabolism is very vigorous, the 20-80 when blood flow during skeletal muscle strenuous exercise in it can reach tranquility doubly has 1350-3000 bar blood capillary therefore normal human's skeletal muscle all has in the domination blood vessel of thicker diameter and every square millimeter of muscle; And the blood of UBM and SIS implantation region regeneration skeletal muscle provides for the blood capillary of invading by surrounding tissue, without obviously domination blood vessel, capillary density are also not enough, and the blood flow that needs when violent the contraction can't be provided; 3. UBM and SIS fail to reflect the labyrinth of natural bone myostroma, and the muscle fiber of regeneration skeletal muscle is arranged undesirable.To induce skeletal muscle function regeneration be a very promising technology to ECM in a word, but still have at present the place that is worth the further investigation corrective measure.
Summary of the invention
The invention is characterized in the acellular matrix that has obtained the full organ of skeletal muscle, has high tissue compatibility, the three-dimensional ultrastructure of skeletal muscle and vascular stroma network and muscle-tendon joint design etc. have been kept, remain with the skeletal muscle specificity extracellular matrix components, more existing various timbering material has better been simulated physics and the biological nature of natural bone myocyte epimatrix.
In a specific embodiments of giving an example, the narration that the present invention is detailed this biological activity skeleton of skeletal muscle WOACM flesh regeneration support material preparation method, comprise obtaining and preparation, pre-process, taking off cell, sterilization preservation, external assessment etc. of raw material skeletal muscle tissue.
The skeletal muscle WOACM initiation material of the present invention's preparation is allosome xenogenesis skeletal muscle, as human body, and pig, the skeletal muscle of Canis familiaris L. etc., preferred skeletal muscle are that blood supplies obvious skeletal muscle, optimum is the skeletal muscle of the independent domination of band blood vessel.Scope of selecting material preferably comprises higher level's blood vessel of skeletal muscle domination blood vessel, and optimum is the major blood vessel that comes from the donor body, can conveniently be used for surgical stapling after taking off cell to corresponding blood vessel.The age of donor, because in the extracellular matrix of young donor skeletal muscle, various short stem cell adhesion compositions, somatomedin etc. are abundanter, contained fatty tissue was less take youth as good.The time of drawing materials should be in dead rear 30 minutes of donor, and preferred version is to draw materials when the position skeletal muscle of drawing materials still possesses effective blood circulation.
The raw material that the present invention relates to prepares to refer to that skeletal muscle preserves perfusion preferably obtaining Shi Yingyou, preferably donor has whole body heparinization in advance, best is that the local liquid of preserving of donor whole body heparinization associating in advance fully pours into, fully the standard of perfusion is to pour into preservation liquid through the domination tremulous pulse, goes out without the courageous and upright liquid stream of thickness in the vein of domination tremulous pulse companion row.After fully pouring into, raw material is freezing, rewarming repeatedly.
Skeletal muscle scope, domination tremulous pulse that the pre-process that the present invention relates to indicates the intubate tremulous pulse domination of true institute have or not anatomic variation, and the accompanying vein of intubate domination tremulous pulse is removed fascia, the fatty tissue on raw material skeletal muscle surface;
the cell technology of taking off that the present invention relates to comprises intubate blood vessel access bioreactor, through domination blood vessel sequence perfusion different disposal liquid, comprise that the enzyme processing is as trypsin, deoxyribonuclease (Deoxyribonuclease, DNase), alpha-galactosidase etc., chemicals comprises ethylenediaminetetraacetic acid (Ethylene diamine tetraacetic acid, EDTA), ethyleneglycol bistetraacetic acid (Ethylene glycol tetraacetic acid, EGTA), sodium lauryl sulphate (Sodium dodecyl sulfate, SDS), Triton X-100 (Triton-X), deoxycholic acid (Deoxycholic acid) etc., said medicine can form kinds of schemes and take off cell for skeletal muscle, between scheme at choice of drug, the drug use order, concentration and action time are upper variant, but congruence, namely realize thoroughly taking off cell in the gentleest mode and keep to greatest extent bioactive ingredients in the Skeletal Muscle Cell epimatrix simultaneously.
Taking off cell scheme tryptic final concentration used is 0.01～0.25%, the final concentration of EDTA or EGTA is 0.02～0.5%, the final concentration of DNase is 30-50U/ml, the final concentration of alpha-galactosidase is 10-25U/ml, and the final concentration of SDS is 0.01～0.1%, the final concentration of Triton-X is 0.1～1%, the final concentration of Deoxycholate is 0.5～2%.
Take off the 1-3 hour action time of cell scheme trypsin/EDTA used or EGTA, the 0.4-1 hour action time of DNase and alpha-galactosidase, the 4-24 hour action time of the 8-24 hour action time of the 4-24 hour action time of SDS, Triton-X, Deoxycholate.
The disinfection technology that the present invention relates to comprises through domination vascular perfusion peracetic acid (Peroxyacetic acid, PAA)/ethanol (ETOH) mixed liquor or gamma Rays sterilization etc.Wherein the final concentration of PAA (V/V) is 0.1-0.5%, and the final concentration of ethanol (V/V) is 2-10%, the 2-3 hour action time of PAA/ alcohol mixeding liquid.The skeletal muscle WOACM of preparation can select aquation to preserve, or forms the dry body preservation through vacuum lyophilization.
in a specific embodiments of giving an example, the narration that the present invention is detailed detection method and the result of this biological activity skeleton of skeletal muscle WOACM flesh regeneration support material Bioactivity, comprise the histology who detects skeletal muscle WOACM, ultrastructure detects cell free completeness (residual nucleic acid, endotoxin content and biological load such as fungus, viral level, the size of residual nucleic acid chains, the residual cell compound content that takes off), detect the special inhomogeneous existence of skeletal muscle basement membrane such as IV Collagen Type VI, laminin, fibronectins etc. are measured short stem cell and are adhered to, content such as the hyaluronic acid of propagation and differentiation composition, glycosaminoglycans (Glycosaminoglycan, GAGs), heparitin sulfate, various somatomedin such as vascular endothelial cell growth factor (Vascular endothelial growth factor, VEGF), basic fibroblast growth factor (Basic fibroblast growth factor, bFGF), transforming growth factor-beta (transforming growth factor-β, TGF-β) etc.
In a specific embodiments of giving an example, the narration that the present invention is detailed skeletal muscle WOACM than other timbering materials in the advantage aspect short blood vessel and Muscle-derived Stem Cells chemotactic, short blood vessel and Muscle-derived Stem Cells propagation, short Muscle-derived Stem Cells myogenic differentiation and myotube generation.
The prepared skeletal muscle WOACM of the present invention can derive the medical products such as microgranule, fluidisation compositions, gel and bioactive peptide.Particulate product is for to pulverize gained with the dry body after skeletal muscle WOACM vacuum lyophilization through high speed rotating, comprise a plurality of product hierarchies (≤38 μ m, 38-100 μ m, 100-250 μ m), can be used as the covering of skeletal muscle wound surface, the damaged filler of skeletal muscle etc.The fluidisation compositions is that skeletal muscle WOACM microgranule is used the pepsin digestion gained in sour environment.Gel products is that homogeneous (uniform) fluid compositions reconcentration, neutralization are processed gained, comprises the variable concentrations grade, can be used as that the skeletal muscle wound surface covers, injection filling skeletal muscle is damaged, treatment muscular dystrophy etc.The technology for preparing above-mentioned derived product is all well-known to those skilled in the art.
The preparation of the skeletal muscle WOACM bioactive peptide that the present invention relates to: with skeletal muscle WOACM fluidisation compositions through ultrahigh speed centrifugal (〉=13000rpm) after, supernatant vacuum lyophilization, the gained dry body is the bioactive peptide of skeletal muscle WOACM, and testing in vitro confirms that the ability of its short skeletal myoblast chemotactic, propagation and differentiation is more efficient far beyond skeletal muscle WOACM fluidisation compositions.
By the description of this paper, other aspect of the present invention and feature and advantage are apparent for those of ordinary skills.
Description of drawings
Fig. 1 is domination vascular anatomy and the scope of selecting material exemplary plot of rectus abdominis m. under pig;
Fig. 2 is original skeletal muscle and by the skeletal muscle schematic diagram of this piece muscle preparation;
Fig. 3 is skeletal muscle WOACM structural integrity, remains with the exemplary plot of blood vessel network;
Fig. 4 is the transverse section of natural bone flesh and skeletal muscle WOACM, along the scanning electron microscope (SEM) photograph of macropinacoid;
Fig. 5 is the transverse section scanning electron microscope (SEM) photograph of blood capillary network of the visible reservation of skeletal muscle WOACM;
Fig. 6 is HE dyeing, basement membrane proteins immunohistochemical staining (200X) exemplary plot of natural bone flesh and skeletal muscle WOACM transverse section;
Fig. 7 is that natural bone flesh and skeletal muscle WOACM are along HE dyeing, basement membrane proteins immunohistochemical staining (200X) exemplary plot of macropinacoid.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Find after deliberation, skeletal muscle ECM is more conducive to skeletal muscle regeneration, and wherein skeletal muscle WOACM has best theory advantage.More and more studies show that in recent years, the ECM of particular organization all has the unique composition and structure of this tissue, the composition and structure that same skeletal muscle ECM also possesses skeletal muscle specificity is high IV Collagen Type VI content 1., mainly be present in the skeletal muscle basement membrane, be beneficial to sarcoplast and adhere to, move and activate; 2. complicated, three-dimensional ultrastructure that have start-up performance, comprise the endomysium tubular construction that is arranged in parallel, remaining nerve and vascular access, can promote myoblastic adjustment and fusion, and the angiogenic growth that excites nerve; 3. be rich in somatomedin, hyaluronic acid, laminin，LN, heparin sulfate, S-PG etc., biocompatibility is splendid; 4. catabolite has mitosis and the chemotaxis of strong short muscle stem cell, and the microenvironment of these catabolites formation, and is extremely important to the vigor of keeping myocyte and satellite cell.Therefore, skeletal muscle ECM should be more suitable in rebuilding skeletal muscle than other materials, and skeletal muscle ECM reparation skeletal muscle also should have better regeneration induction effect when damaged.
Preparation skeletal muscle ECM is the difficult problem in organizational project and regenerative medicine field always.It has been generally acknowledged that structure of skeletal muscles is fine and close, there is multiple connective tissue to separate between tissue, take off cell liquid in wherein being difficult to disperse, therefore the report that only has so far the ECM of rat extremity fritter skeletal muscle ECM and Animal muscles thin slice to be successfully prepared, but these skeletal muscle ECM can not be damaged for the skeletal muscle on a large scale of repairing clinically.2008 international top medical journals " Nature Medicine " at first reported a revolutionary technology, perfusion is taken off the extracellular matrix that cell prepares whole organ and is used for building corresponding organizational project organ.These methods of application such as American scholar Ott HC are prepared the full organ acellular matrix of rat heart, successfully obtain an artificial heart after the inoculation stem cell in vitro builds 8 days, its blood-pumping function reach the Normal Adult Rat heart 2%, 16 all tire rat hearts 25%.Be subject to technique and inspire, people have successively obtained the full organ acellular matrix of the internal organs such as liver, kidney, lung, and related article all is published in the top periodicals such as " Science ", " Hepatology ".Meanwhile, the preparation work of skeletal muscle WOACM is also being carried out at internationally recognizable organizational project center always, because in theory, the various skeletal muscle regeneration platforms that skeletal muscle WOACM has reported at present both at home and abroad, comprise inorganic bio such as soluble phosphate glass fibre support etc., biological degradable synthesized polymer material such as polyglycolic acid, polylactic acid and and all kinds of ester monomer copolymer such as polylactic acid-glycollic acid etc., natural degradable biomaterial such as chitosan, collagen, SIS and UBM etc. have many unrivaled advantages:
A. skeletal muscle WOACM has kept that the three-dimensional skeletal muscle complexity, that have start-up performance is arranged in parallel, periodic endomysium (basement membrane) tubular construction, such ultrastructure still can not manually be imitated at present, be beneficial to especially Muscle-derived Stem Cells directional migration, extension and fusion, also can guide the direction of newborn myotube;
B. skeletal muscle WOACM remains with bioactive ingredients (keeping the bio signal of Muscle-derived Stem Cells activity): comprise that the special composition of skeletal muscle basement membrane (IV Collagen Type VI, laminin, fibronectin), short stem cell adhere to, breed and differentiation composition (hyaluronic acid, glycosaminoglycans, heparitin sulfate and somatomedin such as bFGF, TGF-β 1, IGF, VEGF etc.);
C. skeletal muscle WOACM has kept the complete vascular stroma network of skeletal muscle, comprises terminal artery, vein and blood capillary system; Be used for systematicness inoculation stem cell can guarantee stem cell be evenly distributed in whole substrate and the cell of all inoculations around blood vessel in 150-200 μ m, can good survival; In addition, can be stem cell and continue to provide nutrition if the vascular stroma network can be communicated to the blood circulation of receptor;
D. skeletal muscle WOACM has kept the substrate of main supply blood vessel, and the major diameter supply blood vessel that makes new advances of having an opportunity to regenerate is arranged newborn skeletal muscle becomes global function skeletal muscle;
E. skeletal muscle WOACM has kept muscle-tendon joint design, can directly conduct mechanics;
F. skeletal muscle WOACM has certain mechanical strength and toughness;
G. skeletal muscle ECM more easily realizes the neuralization again of newborn skeletal muscle than other materials.Studies show that, when slight Skeletal muscle injury body is realized self-regeneration, surpass still regenerate original position (satellite cell tabernacle) on basement membrane and endomysium of 95% neuromuscular junction, and skeletal muscle WOACM has kept the composition and structure of muscle satellite cell tabernacle.
The obtaining and prepare of raw material (rectus abdominis m. under the pig stomach wall): donor is female Yorkshaire pig in 16-20 age in week, body weight 25-30kg, in advance through peripheral vein whole body heparinization (10000U/ only), employing is from xiphoid-process until the median abdominal incision of the nearly anus of lower abdomen, cut hunter's line to preperitoneal space, push peritoneum open to cephalad direction, structure after the exposure peritoneum comprises abdomen initiatively vein, the total blood vessel of ilium, the outer blood vessel of ilium.Cut external iliac artery, insert the 14-20G sleeve pipe to inferior epigastric artery, charge pump drives the normal saline that injects heparinization (1-5U/ml) in the inferior epigastric artery, keeps Intraarterial pressure in live body normal arterial pressure level (100-120mmHg).Fully perfusion is until go out without dense thick courageous and upright liquid stream in inferior epigastric vein.Continuation blood vessel outside ilium exposes pudendum stomach wall blood vessel and does (this blood vessel is done and is comprised of three blood vessels: inferior epigastric artery, upper abdomen tail shallow artery and external pudendal arteries) and femoral artery (Fig. 1 is left).Peel off theca of rectus abdominis muscle along the rectus abdominis m. front surface, peel off sheath (above arcuate line) after transverse fascia (arcuate line below) and rectus abdominis m. along the rear surface, until umbilicus level (the 3rd or the 4th tendon is drawn), feature is dark perforator artery (Deep inferior epigastric perforator, DIEP) under visible stomach wall.Cut off rectus abdominis m. tendon and pubis joint along pubic arch surface, cut off the outer arteriovenous of ilium, burst arteriovenous, the outer arteriovenous of pudendum and the shallow arteriovenous of upper abdomen tail, cut off rectus abdominis m. along the umbilicus level, complete the obtaining and preparing of lower rectus abdominis m. (Fig. 1 is right).
Pre-process: under the pig that obtains, rectus abdominis m. is placed in the perfusion reactor, and the external iliac artery intubate is communicated with charge pump, the phosphate buffer (PBS) of low pressure (approximately 60mmHg) perfusion 1X.Use the clear and definite inferior epigastric artery domination of dyestuff scope, checking has the abnormal vascular of unavailability lower abdomen rectus, if having, need put pipe and enter this blood vessel for perfusion (Fig. 2 is left).Further remove the fatty tissue on muscular tissue surface, ligation external iliac artery all branches except inferior epigastric artery comprise upper abdomen tail shallow artery, external pudendal arteries and femoral artery, guarantee to flow into inferior epigastric artery fully through the external iliac artery infusion liquid.Ligation external iliac vein all branches except inferior epigastric vein (having 2) comprise upper abdomen tail superficial vein (2), external pudendal veins and femoral vein, insert sleeve pipe in external iliac vein.
Taking off cell processes: with the outer arteriovenous sleeve pipe access of ilium bioreactor, note during perfusion specimen must be immersed in simultaneously with the same liquid of infusion liquid in.Subordinate list 1 and 2 is that 2 kinds of commonly using take off the cell scheme, and wherein 1 operating time of scheme is longer than scheme 2, but realizes that more easily ultra-large volume is (as the cell that takes off greater than 50 * 40 * 5cm) raw material skeletal muscle.Skeletal muscle is transparence after taking off cell, can clear demonstration blood vessel, neutral net (Fig. 2 is right) in it, its structural integrity, no liquid seepage (Fig. 3) are confirmed in the dyeing perfusion.
Sterilization and preservation: after the 0.1%PAA/4%ETOH perfusion, the liquid of all perfusions comprises deionized water, is sterilized liquid.The final skeletal muscle WOACM that obtains is stored in tissue preservation liquid, the gamma Rays sterilization.Preserve liquid composition lactobionic acid 100mmol, potassium hydrogen phosphate 25mmol, magnesium sulfate 5mmol, sodium hydroxide (5mmol/L) 5ml, potassium hydroxide (5mmol/L) 20ml, sucrose 60mmol, pH7.45 ± 0.10, osmotic pressure (310 ± 10) mOsm/L.
The ultrastructure of skeletal muscle WOACM: HE dyeing and scanning electron microscope confirm that skeletal muscle WOACM has kept that natural bone myocyte epimatrix three-dimensional is arranged in parallel, periodically epimysium, perimysium and endomysium ultrastructure, the skeletal muscle basement membrane components is attached at endomysium inner face (Fig. 4), and such structure is adhesion, migration, propagation, differentiation and the fusion that is beneficial to Muscle-derived Stem Cells most.The blood capillary network that as seen keeps clearly (2 terminal veins of terminal artery and companion's row, Fig. 5).
Taking off the cell completeness measures: histological stain has no the nucleus structure, and DAPI dyes negative.Extract all DNA in skeletal muscle WOACM, use the Picogreen method to measure the content of residual DNA in skeletal muscle WOACM dry weight lower than 50ng/mg.The length 200-500bp of residual DNA chain.
Biological activity determination: measure through the ELISA method, in skeletal muscle WOACM dry weight, GAGs content is that 645 ± 40ng/mg, VEGF content are 186 ± 22ng/mg, and bFGF content is 1375 ± 230ng/mg, and TGF-β content is 293 ± 8ng/mg.The dyeing of skeletal muscle basement membrane main component protein immunization group confirms to contain IV Collagen Type VI, laminin, fibronectin in skeletal muscle WOACM.
skeletal muscle WOACM is than other ECM and synthesized polymer material more chemotactic blood vessel and Muscle-derived Stem Cells: prepared skeletal muscle WOACM fluidisation compositions is after neutralization, with variable concentrations (1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 5 μ g/ml) comparison and SIS, UBM, PLGA is to skeletal myoblast, the chemotaxis of MJK/PJK (Boyden chamber method), found that skeletal muscle WOACM has best short stem cell chemotaxis, and reach the best use of when concentration 25 μ g/ml.
Skeletal muscle WOACM more promotes blood vessel and Muscle-derived Stem Cells propagation than other ECM and synthesized polymer material: use the BrdU method to detect skeletal muscle WOACM, SIS, UBM, the PLGA of variable concentrations (1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 5 μ g/ml) to the proliferation of skeletal myoblast, MJK/PJK, found that skeletal muscle WOACM has best short stem cells hyperplasia effect, and reach the best use of when concentration 50 μ g/ml.
Skeletal muscle WOACM more promotes blood vessel and Muscle-derived Stem Cells differentiation than other ECM and synthesized polymer material: inoculation 5 * 10
5Skeletal myoblast to 1 * 1cm skeletal muscle WOACM thin slice, SIS, UBM, PLGA rack surface, use full culture fluid (DMEM+10% hyclone) to cultivate and use inducing culture liquid (DMEM+10% horse serum) cultivation 3 days after 7 days instead, relatively the differentiation of skeletal myoblast and flesh generate, detect the expression (MyoD of skeletal muscle regulatory factor, Myogenin, MRF4, Myf5), laser confocal microscope detects amount, size and the order of newborn myotube.Found that the surperficial sarcoplast of skeletal muscle WOACM the most easily to form newborn myotube, and newborn myotube is arranged the most orderly.
Residual chemical is measured: the skeletal muscle WOACM that takes off 1 preparation of cell scheme adopts the sodium lauryl sulphate in the methylene blue Bound residues, the Picogreen method is measured absorbance, measure residual sodium lauryl sulphate in skeletal muscle WOACM dry weight content lower than 100ng/mg, Triton-X do not detected residual; Take off and Triton-X do not detected in the skeletal muscle WOACM of cell scheme 2 preparation and deoxycholic acid is residual.
Application
The skeletal muscle WOACM of the present invention preparation and the application of derivative medical product thereof comprise for organizational project and regenerative medicine field external structure, internal regeneration skeletal muscle, the skeletal muscle that can repair various scopes is damaged to be comprised and covers the skeletal muscle wound surface, fills that flesh is damaged, injection for curing muscular dystrophy, dissects and repair damagedly on a large scale, also can be used for the treatment of myocardial infarction.
Above disclosed be only the application's a specific embodiment, but the application is not limited thereto, the changes that any person skilled in the art can think of all should drop in the application's protection domain.
Subordinate list, description of drawings
Table 1. takes off cell scheme 1:
| Perfusion fluid | Perfused vessel and time |
| 0.02%Trypsin/0.05%EGTA | Tremulous pulse 1.75h+ vein 0.25h |
| Deionized water, 2X PBS | Tremulous pulse, each 0.5h |
| 0.01%SDS | Tremulous pulse 11h+ vein 1h |
| Deionized water | Tremulous pulse, 0.5h |
| 0.5%Triton?X-100 | Tremulous pulse 11h+ vein 1h |
| Deionized water | Tremulous pulse, 24h |
| 0.1%PAA/4%ETOH | Tremulous pulse, 2h |
| Deionized water | Tremulous pulse, 0.5h |
| The DNase/ alpha-galactosidase | Tremulous pulse, 0.5h |
| Deionized water | Tremulous pulse, 72h |
Table 2. takes off cell scheme 2
| Perfusion fluid | Perfused vessel and time |
| 0.02%Trypsin/0.05%EGTA | Tremulous pulse 1.75h+ vein 0.25h |
| Deionized water, 2X PBS | Tremulous pulse, each 0.5h |
| 0.5% ursodesoxycholic acid | Tremulous pulse 11h+ vein 1h |
| Deionized water | Tremulous pulse, 0.5h |
| 0.5%Triton?X-100 | Tremulous pulse 20h+ vein 4h |
| Deionized water | Tremulous pulse, 0.5h |
| 0.5% ursodesoxycholic acid | Tremulous pulse 5.5h+ vein 0.5h |
| Deionized water | Tremulous pulse, 24h |
| 0.1%PAA/4%ETOH | Tremulous pulse, 2h |
| Deionized water | Tremulous pulse, 0.5h |
| The DNase/ alpha-galactosidase | Tremulous pulse, 0.5h |
| Deionized water | Tremulous pulse, 12h |
Claims (7)
1. the full organ acellular matrix of skeletal muscle WOACM, a kind of novel skeletal muscle regeneration support and platform is characterized in that,
Skeletal muscle WOACM has kept that the three-dimensional skeletal muscle complexity, that have start-up performance is arranged in parallel, periodic endomysium tubular construction, and this ultrastructure is beneficial to Muscle-derived Stem Cells directional migration, extension and fusion especially, also can guide the direction of newborn myotube;
Skeletal muscle WOACM has kept the complete vascular stroma network of skeletal muscle, comprises terminal artery, vein and blood capillary system;
Skeletal muscle WOACM remains with the bioactive ingredients of the bio signal of keeping the Muscle-derived Stem Cells activity: comprise the special composition of skeletal muscle basement membrane as IV Collagen Type VI, laminin, fibronectin, as hyaluronic acid, glycosaminoglycans, heparitin sulfate adhere to interior short stem cell, propagation and differentiation composition, as vascular endothelial cell growth factor, basic fibroblast growth factor, transforming growth factor-beta at interior somatomedin;
Skeletal muscle WOACM remains with muscle-tendon joint design, can directly conduct mechanics;
Skeletal muscle WOACM has certain mechanical strength and toughness;
Skeletal muscle WOACM more easily realizes the neuralization again of newborn skeletal muscle than other materials;
It is thorough that skeletal muscle WOACM takes off cell, and utmost point low endotoxin, nucleic acid content do not carry fungus, virus at interior biological load, after implanting without obvious immunological rejection, do not spread disease, but allosome xenotransplantation, do not produce ethics morals problem.
2. the derivative medical product of skeletal muscle WOACM comprises particulate product, fluidisation compositions, gel and bioactive peptide etc.; Skeletal muscle WOACM, together with its various medical products that derive, can be used as external construct in vitro and regeneration skeletal muscle, the skeletal muscle that is applicable to repair body each position, various scopes is damaged, comprise cover that skeletal muscle wound surface, filling skeletal muscle are damaged, injection for curing muscular dystrophy etc., particularly be expected to solve at present clinically still very thorny, skeletal muscle defect repair and an anathrepsis difficult problem on a large scale.
3. the technology of preparing of skeletal muscle WOACM according to claim 1, comprise and raw-materially obtain and preparation, pre-process, take off that cell technology, sterilization are preserved, external assessment technology, the technology of the present invention is applicable to prepare the skeletal muscle WOACM at all biologies, each position, as rectus abdominis m., the latissimus dorsi m. of human body, pig.
4. raw-material obtaining and preparation techniques according to claim 3, its standard are to guarantee that skeletal muscle is fully poured into, blood constituent is discharged from and can not condense in the little blood vessel of skeletal muscle to cause microcirculation blockage.
5. the cell technology of taking off according to claim 3, comprise the access bioreactor, sequence is poured into enzyme and chemicals and auxiliary reagent to the domination blood vessel of skeletal muscle according to listed concentration, and standard is to realize thoroughly taking off cell, keep to greatest extent bioactive ingredients in the Skeletal Muscle Cell epimatrix simultaneously with the gentleest scheme.
6. disinfection technology according to claim 3 comprises sterilised liq perfusion, radiation sterilization; Described Techniques of preserving comprises that aquation is preserved, dry body is preserved.
7. assessment technique according to claim 3, comprise the histology, the ultrastructure that detect skeletal muscle WOACM, detect the length of residual nucleic acid, endotoxin content and residual nucleic acid chains, measure biological load such as fungus, virus and the residual cell compound content that takes off, the existence and the short stem cell that detect skeletal muscle basement membrane specific component adhere to, breed and break up the content of composition.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104383601A (en) * | 2014-10-30 | 2015-03-04 | 上海交通大学医学院附属第九人民医院 | Skeletal muscle acellular matrix biological patch and preparation method thereof |
| CN104740684A (en) * | 2015-03-03 | 2015-07-01 | 上海市闸北区中心医院 | Canine uterus whole-organ acellular matrix and preparation method as well as application of canine uterus whole-organ acellular matrix |
| CN104971380A (en) * | 2014-04-11 | 2015-10-14 | 烟台隽秀生物科技有限公司 | Acellular matrix repairing gel and new method for preparing the same |
| CN108030959A (en) * | 2017-09-12 | 2018-05-15 | 浙江大学 | The muscle of sustained release IGF-1 growth factors takes off cell material and preparation method thereof |
| CN108514657A (en) * | 2018-04-04 | 2018-09-11 | 浙江大学 | The muscle for being sustained IGF-1 growth factors takes off the preparation method of cell material |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018140855A1 (en) | 2017-01-30 | 2018-08-02 | Lifecell Corporation | Devices including muscle matrix and methods of production and use |
| CN108465125A (en) * | 2018-04-04 | 2018-08-31 | 浙江大学 | The tendon composite muscle in natural tissues source takes off the preparation method of cell material |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101272815A (en) * | 2005-08-26 | 2008-09-24 | 明尼苏达大学董事会 | Decellularization and recellularization of organs and tissues |
| WO2010039823A2 (en) * | 2008-09-30 | 2010-04-08 | The Regents Of The University Of California | Compositions and methods for tissue repair with extracellular matrices |
-
2013
- 2013-02-26 CN CN2013100605866A patent/CN103127550A/en active Pending
-
2014
- 2014-02-26 CN CN201410067736.0A patent/CN103805555B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101272815A (en) * | 2005-08-26 | 2008-09-24 | 明尼苏达大学董事会 | Decellularization and recellularization of organs and tissues |
| WO2010039823A2 (en) * | 2008-09-30 | 2010-04-08 | The Regents Of The University Of California | Compositions and methods for tissue repair with extracellular matrices |
| CN102227225A (en) * | 2008-09-30 | 2011-10-26 | 加利福尼亚大学董事会 | Compositions and methods for tissue repair with extracellular matrices |
Non-Patent Citations (3)
| Title |
|---|
| BENJAMIN等: "The promotion of a functional fibrosis in skeletal muscle with volumetric muscle loss injury following the transplantation of muscle-ECM", 《BIOMATERIALS》, vol. 34, no. 13, 4 February 2013 (2013-02-04), pages 3324 - 3335 * |
| 卿泉等: "大鼠骨骼肌脱细胞处理方法的优化研究", 《中国修复重建外科杂志》, vol. 23, no. 7, 31 July 2009 (2009-07-31), pages 836 - 839 * |
| 田春祥等: "骨骼肌无细胞基质的制备及其生物相容性研究", 《中国修复重建外科杂志》, vol. 26, no. 6, 30 June 2012 (2012-06-30), pages 749 - 754 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104971380A (en) * | 2014-04-11 | 2015-10-14 | 烟台隽秀生物科技有限公司 | Acellular matrix repairing gel and new method for preparing the same |
| CN104383601A (en) * | 2014-10-30 | 2015-03-04 | 上海交通大学医学院附属第九人民医院 | Skeletal muscle acellular matrix biological patch and preparation method thereof |
| CN104740684A (en) * | 2015-03-03 | 2015-07-01 | 上海市闸北区中心医院 | Canine uterus whole-organ acellular matrix and preparation method as well as application of canine uterus whole-organ acellular matrix |
| CN108030959A (en) * | 2017-09-12 | 2018-05-15 | 浙江大学 | The muscle of sustained release IGF-1 growth factors takes off cell material and preparation method thereof |
| CN108514657A (en) * | 2018-04-04 | 2018-09-11 | 浙江大学 | The muscle for being sustained IGF-1 growth factors takes off the preparation method of cell material |
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| CN103805555A (en) | 2014-05-21 |
| CN103805555B (en) | 2015-12-30 |
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