CN101695583B - Granular biological material for tissue repair and preparation method thereof - Google Patents
Granular biological material for tissue repair and preparation method thereof Download PDFInfo
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- CN101695583B CN101695583B CN 200910191251 CN200910191251A CN101695583B CN 101695583 B CN101695583 B CN 101695583B CN 200910191251 CN200910191251 CN 200910191251 CN 200910191251 A CN200910191251 A CN 200910191251A CN 101695583 B CN101695583 B CN 101695583B
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- granular biological
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
Abstract
The invention provides a granular biological material for tissue repair and a preparation method thereof. The method comprises the steps of: firstly, freeze-drying a film-like biological material prepared from one or combination of at least two of acellular tissue matrices, collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyglycollic acid and alginate; then cutting the film-like biological material to be strip-shaped under set parameters at normal temperature; and finally, cutting the film-like biological material into particles with the particle size range of between 60 and 6,000mu m, and packaging and disinfecting the granular biological material. The granular biological material and the method have the advantages that: preparation conditions are mild, the preparation method is simple, prepared products have regular shapes, and the particle size distribution concentration ratio of the particles is more than or equal to 85 percent; and the granular biological material can be directly used for repairing tissues or organs by adopting methods of injection, spraying, spreading and filling, can also be used after carrying corresponding tissue cells or related molecules for promoting the healing and regeneration of the tissues, and can meet the need on the repair of various tissues or organs.
Description
Technical field
The invention belongs to the prosthetic material or the prosthese lining material of what field of tissue engineering technology, particularly a kind of granular biological material that is used for tissue repair and preparation method thereof.
Background technology
In the skin tissue recovering field, how to utilize the limited normal skin tissue of patient, enlarge tissue transplantation's scope as far as possible, be to need improved major issue in the large skin defect repairing and treating.With normal skin tissue or prosthese lining material granulating, adopt the mode that sprays, sows to achieve the above object, be widely used in large-area burns patient's treatment at present.The granulating tissue biomaterial that injectable maybe can be filled also is widely used in cosmetic plastic surgery, neurosurgery, Urology Surgery, Oral and Maxillofacial Surgery field, in the hope of reach best repairing effect with minimally invasive.At present; Tissue repair is also being sought suitable gritty texture's biomaterial as carrier for tissue engineering seed cell with the focus of regeneration research; Organize, the treatment of the cell transplantation of organ, carry out corresponding tissue repair etc. such as the cell of skin, nerve, cartilage and cardiac muscle or stem cell are carried on gritty texture's biomaterial.It is thus clear that, how to prepare be fit to different tissues reparation needs, tissue biomaterial granule of uniform size is the focus of R&D organization reparation and regrown material always.
With the method for particles of bioglassization, the prior art report of making known publicly is fewer.U.S. LifeCell company have one April 15 in 2008 the invention disclosed patent, publication number is US7,358; 284, its preparation method is made a summary as follows: adopt Zimmer netted skin preparing device in transformation back to grind exsiccant AlloDerm and shred, the employing refiner rubs slice under ultralow temperature then; Be twisted into and use different screen clothes at low temperatures behind the granule it is separated; At last the granule lyophilizing is handled packing, sterilization; The distribution of particles concentration degree of its preparation is 68%; Particle size range is 58 μ m ~ 593 μ m [Sclafani AP; Romo T, Jacono A.Evaluation of acellular dermal graftin sheet (AlloDerm) and injectable (micronized AlloDerm) forms forsoft tissue augmentation:clinical observations and histologicalanalysis.Arch Facial Plast Surg 2000; 2:130-6.].Also have one on June 11st, 2008 disclosed CN101195044A, it is that acellular dermal matrix is ground into granule behind freezing, multigelation, the size after sieving is 50 μ m～500 μ m.But above-mentioned prior art still exists following shortcoming: (1) preparation condition is harsh, needs ultralow temperature and low temperature; (2) the preparation manipulation step is various, needs through freezing, multigelation; (3) the granule profile irregularity of preparation; (4) even the granule of preparation sieves, its particle size distribution is not still concentrated; (5) the too small granule (less than 52 microns) of particle diameter will produce damage to the immunocyte of body.
Summary of the invention
The object of the invention is exactly the defective that exists to prior art, and a kind of granular biological material that is used for tissue repair and preparation method thereof is provided, and makes it the preparation condition gentleness; Method for preparing is simple, and the particle shape of preparation is regular, and the particle size range that can prepare is big; The centralized particle diameter of single specification; Be not only applicable to direct application, also can load respective organization cell or promote the correlation molecule of tissue regeneration, to satisfy the needs that multiple tissue or organ are repaired.
General plotting of the present invention is: the controllability of utilizing the machine cuts parameter; Through being set, different cutting parameters controls prepared particulate size; Thereby make that the particle size range for preparing a certain sized particles is controlled, it is concentrated relatively to distribute, and the grain diameter scope that can cut is big; And then realize the membrane-like biomaterial is directly cut into granular biological material under room temperature, satisfy the needs that multiple tissue or organ are repaired.
For realizing the object of the invention, the technical scheme of being taked is following.
A kind of granular biological material that is used for tissue repair, raw material are to take off in cell tissue substrate (for example acellular dermal support), collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyglycolic acid, the alginate any or at least two kinds of combinations and the membrane-like biomaterials of preparation; The form of finished particle is regular, and profile is cube or cuboid; The particle grain size scope is 60 μ m～6000 μ m; Particle size distribution concentration degree >=85% of single specification.
Its method for preparing is prepared three step processes and is formed by lyophilizing, the preparation of fine strip shape biomaterial, the granular biological material of membrane-like biomaterial.
1) lyophilizing of membrane-like biomaterial
The membrane-like biomaterial is placed freeze dryer, is 0.04mbar～0.4mbar in vacuum, and temperature is lyophilizing 3 hours～12 hours under-30 ℃～-70 ℃ conditions, makes it remove moisture, bone dry.
2) fine strip shape biomaterial preparation
Under the room temperature, in the scope of 60 μ m～6000 μ m, the width cutting parameter is set, the membrane-like biomaterial is cut into the fine strip shape biomaterial of set width parameter.
3) granular biological material preparation
Under the room temperature, in the scope of 60 μ m～6000 μ m, the grain graininess cutting parameter is set, the fine strip shape biomaterial is cut into the granular biological material of set grain graininess parameter; Again with the granular biological material that obtains be distributed into 1g～100g directly be soaked in preserve in 75% ethanol or the PVC evacuation after pack; With packing behind 10kGy～25kGy cobalt 60 radiation treatment preservation or the PVC evacuation, oxirane disinfection is preserved at last.
Granular biological material of the present invention and preparation method thereof has the following advantages: the one, and the cutting condition is a room temperature, need not ultralow temperature or low temperature; The 2nd, method is easy and simple to handle, is convenient to production line operation; The 3rd, the product form that obtains is regular, and profile is cube or cuboid granule; The 4th, need not to sieve particle size distribution concentration degree >=85% of same size; The 5th, the particle grain size scope is 60 μ m ~ 6000 μ m, can mix use between the particles of different sizes; The 6th, particles of different sizes can adopt injection, sprays, sows, the filling method is applied to tissue or the organ reparation; The 7th, raw material sources are extensive, can be to take off in cell tissue substrate, collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyglycolic acid, the alginate any or at least two kinds of combinations and the membrane-like biomaterials of preparation.
The granular biological material of the present invention's preparation is suitable for direct application, also can or promote at load respective organization cell to use behind organization healing and the regenerated correlation molecule, can satisfy the needs that multiple tissue or organ are repaired.
Description of drawings
Fig. 1 is the particulate gross examination of skeletal muscle photo of 1.2mm specification acellular dermal support.
Fig. 2 is a 0.7mm specification acellular dermal support particle size distribution testing result.
Fig. 3 is a 0.2mm specification acellular dermal support particle size distribution testing result.
Fig. 4 is 3 hours a microscopically photo (400 times) behind the inoculation people keratinocyte on the acellular dermal support granule
Fig. 5 is that acellular dermal support granule cograft detects from the biomechanics characteristic of body tomography skin (STSG+MADM) postoperative 20 all wound healing skins.
The specific embodiment
Enumerate below and use a plurality of embodiments of acellular dermal support, further specify granular biological material of the present invention and preparation method thereof as the feedstock production granular biological material.It is emphasized that the present invention is not limited to cited embodiment.
Specification is that the particulate concrete method for preparing of the acellular dermal support of 1.2mm is following:
(1) acellular dermal support lyophilizing: the acellular dermal of preparation is propped up and is placed in the freeze dryer, vacuum 0.1mbar, lyophilizing 6 hours, temperature-45 ℃;
(2) fine strip shape acellular dermal support preparation: it is 1.2mm that cutting parameter is set, and the acellular dermal support after the lyophilizing is cut into slice (width: 1.2mm) shape biomaterial;
(3) graininess acellular dermal support preparation: it is 1.2mm that cutting parameter is set; With the width of preparation is that the fine strip shape acellular dermal support of 1.2mm cuts into granule (specification: 1.2mm); Prepared particle shape is regular, and size is even, and mean diameter is 1261 μ m (see figure 1)s;
(4) packing, sterilization: the acellular dermal support granule of preparation is distributed into 1g, directly is soaked in 75% ethanol and preserves.
Specification is that the particulate concrete method for preparing of the acellular dermal support of 0.7mm is following:
(1) acellular dermal support lyophilizing: the acellular dermal of preparation is propped up and is placed in the freeze dryer, vacuum 0.1mbar, lyophilizing 6 hours, temperature-55 ℃;
(2) fine strip shape acellular dermal support preparation: it is 0.7mm that cutting parameter is set, and the acellular dermal support after the lyophilizing is cut into slice (width: 0.7mm) shape biomaterial;
(3) graininess acellular dermal support preparation: it is 0.7mm that cutting parameter is set; With the width of preparation is that the fine strip shape acellular dermal support of 0.7mm cuts into granule (specification: 0.7mm); Prepared particle shape is regular, and size is even, and profile is nearly cubic;
(4) packing, sterilization: the acellular dermal support granule of preparation is distributed into 2g, packs behind the PVC evacuation, and oxirane disinfection is preserved;
(5) the particulate mean diameter of Particle Size Analyzer detection acellular dermal support is 875 μ m, and the distribution of particles concentration degree in 479 μ m～1096 μ m particle size range is 90.93% (see figure 2).The result proves that prepared acellular dermal grain diameter span scope is little, and the degree of distribution high concentration.
Embodiment 3:
Specification is that the particulate concrete method for preparing of the acellular dermal support of 0.2mm is following:
(1) acellular dermal support lyophilizing: the acellular dermal of preparation is propped up and is placed in the freeze dryer, vacuum 0.1mbar, lyophilizing 6 hours, temperature-50 ℃;
(2) fine strip shape acellular dermal support preparation: it is 0.2mm that cutting parameter is set, and the acellular dermal support after the lyophilizing is cut into slice (width: 0.2mm) shape biomaterial;
(3) graininess acellular dermal support preparation: it is 0.2mm that cutting parameter is set, and is that the fine strip shape acellular dermal support of 0.2mm cuts into granule (specification: 0.2mm) with the width of preparation;
(4) packing, sterilization: the acellular dermal support granule of preparation is distributed into 2g, packs behind the PVC evacuation, and oxirane disinfection is preserved;
(5) the particulate mean diameter of Particle Size Analyzer detection acellular dermal support is 323 μ m, and the distribution of particles concentration degree in 182 μ m～417 μ m particle size range is 87.86% (see figure 3).The result proves that prepared grain diameter span scope is significantly less than the particulate 58 μ m of AlloDerm ~ 593 μ m, and the distribution of particles concentration degree is higher than nearly 20 percentage points of AlloDerm granule (68%).
Embodiment 4:
The particulate preparation of load people keratinocyte acellular dermal support, the concrete operations step is following:
(1) the particulate preparation of acellular dermal support:, soak the back with normal saline and clean 3 times with the acellular dermal support granule 20mg that preserves.Granule is placed the aseptic centrifuge tube of 1.5ml, add cell culture fluid 1ml and soak that to discard culture fluid after 6 hours subsequent use.
(2) preparation of people's keratinocyte: under the aseptic condition, people's keratinocyte of cultivating is blown and beaten behind 37 ℃ of digestion 5min with 0.25% trypsin; The medium centrifugal 1000rpm that will contain people's keratinocyte, 5min; Supernatant discarded; Mixing cell behind the adding culture fluid, counting.
(3) the particulate cultivation of load people keratinocyte acellular dermal support: add and contain 2 * 10
6The culture fluid 0.5ml of individual/ml people's keratinocyte and subsequent use 20mg particulate vector are mixed in the aseptic centrifuge tube of 5ml, in 37 ℃ of 5%CO
2Incubator leaves standstill hatches 3 hours.
(4) the particulate microscopically of load people keratinocyte acellular dermal support is observed: discard culture fluid, clean load people keratinocyte acellular dermal support granule 2 times with normal saline.Microscopically is observed visible people's keratinocyte attached on the granule support, shows that the acellular dermal support is easy to load people keratinocyte (see figure 4).
Embodiment 5:
Acellular dermal support granule is used for the experiment of skin injury wound repair in animal, and the concrete operations step is following:
(1) making of skin injury model: (male, 220 ~ 260g) anaesthetize (1% pentobarbital, 40mg/kg, lumbar injection), fixedly the full thickness skin graft of back part center clip 4cm * 6cm the SD rat.
(2) prepare from body tomography skin:, for use with the gauze parcel of PBS solution wetted with processing from body tomography skin (STSG) of 4cm * 6cm from the full pachydermia of body.
(3) the particulate preparation of acellular dermal support: the acellular dermal support granule (specification: 0.5mm, area expansion ratio is 2: 1) that will be soaked in 75% ethanol places the funnel that is added with filter paper, with the flushing of PBS solution for several times, and flush away ethanol.
(4) from body tomography skin (STSG) and the particulate cograft of acellular dermal support: on the evengranular corium face of cleaning of sowing at STSG of acellular dermal support, then with its flap coverage, skin graft Edge Distance suture 1mm～2mm during stitching.The wrapping of wound surface postoperative oil yarn, fixing (2 weeks of binder).
(5) postoperative 20 all clips its biomechanics characteristic (see figure 5) of wound surface skin detection that healed; The result shows; With do not add that acellular dermal is particulate to be compared from body tomography skin grafting dermepenthesis rat (STSG), the compound wound surface skin that heals from body tomography skin grafting dermepenthesis rat (STSG+MADM) of acellular dermal granule has higher skin stretch intensity.
Claims (2)
1. granular biological material that is used for tissue repair is characterized in that: it is that raw material makes finished particle by following three steps with the membrane-like biomaterial:
1) lyophilizing of membrane-like biomaterial
The membrane-like biomaterial is placed freeze dryer, is 0.04 mbar～0.4mbar in vacuum, and temperature is lyophilizing 3 hours～12 hours under-30 ℃～-70 ℃ conditions, makes it remove moisture, bone dry;
2) fine strip shape biomaterial preparation
Under the room temperature, in the scope of 60 μ m～6000 μ m, the width cutting parameter is set, the membrane-like biomaterial is cut into the fine strip shape biomaterial of set width parameter;
3) granular biological material preparation
Under the room temperature, in the scope of 60 μ m～6000 μ m, the grain graininess cutting parameter is set, the fine strip shape biomaterial is cut into the granular biological material of set grain graininess parameter; Again with the granular biological material that obtains be distributed into 1g～100g directly be soaked in preserve in 75% ethanol or the PVC evacuation after pack; With packing behind 10kGy～25kGy cobalt 60 radiation treatment preservation or the PVC evacuation, oxirane disinfection is preserved at last;
The form of said finished particle is regular, and profile is cube or cuboid; The particle grain size scope is 60 μ m ~ 6000 μ m; Particle size distribution Ji Zhong Du ≧ 85% of single specification.
2. according to the described granular biological material of claim 1, it is characterized in that said membrane-like biomaterial takes off in cell tissue substrate, collagen, chondroitin sulfate, hyaluronic acid, chitosan, polylactic acid, polyglycolic acid, the alginate any or at least two kinds of combinations and prepares.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910191251 CN101695583B (en) | 2009-10-29 | 2009-10-29 | Granular biological material for tissue repair and preparation method thereof |
| PCT/CN2010/074562 WO2011050622A1 (en) | 2009-10-29 | 2010-06-27 | Granular biomaterial for tissue repairing and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200910191251 CN101695583B (en) | 2009-10-29 | 2009-10-29 | Granular biological material for tissue repair and preparation method thereof |
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| CN101695583A CN101695583A (en) | 2010-04-21 |
| CN101695583B true CN101695583B (en) | 2012-12-26 |
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| WO (1) | WO2011050622A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101695583B (en) * | 2009-10-29 | 2012-12-26 | 中国人民解放军第三军医大学第一附属医院 | Granular biological material for tissue repair and preparation method thereof |
| US9307968B2 (en) * | 2012-06-15 | 2016-04-12 | The University Of Hong Kong | Materials and methods for filling biological cavities and preventing leakage of injected therapeutic agents |
| CA2987045A1 (en) * | 2015-05-27 | 2016-12-01 | Cook Regentec Llc | Sheet-form cell growth scaffold particles and grafts, and methods for same |
| CN112826983A (en) * | 2019-11-22 | 2021-05-25 | 中国科学院大连化学物理研究所 | Heart acellular matrix modified bionic membrane and preparation and application thereof |
| CN111569153A (en) * | 2020-06-04 | 2020-08-25 | 童艳梅 | Application of thin strip-shaped acellular dermal matrix |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6933326B1 (en) * | 1998-06-19 | 2005-08-23 | Lifecell Coporation | Particulate acellular tissue matrix |
| CN101195044A (en) * | 2007-12-29 | 2008-06-11 | 中国人民解放军第四军医大学 | Tissue engineered fine particle tissue and method for preparing the same |
| CN101400738A (en) * | 2006-03-13 | 2009-04-01 | 纳图林有限公司 | Collagen powder and collagen-based thermoplastic composition for preparing conformed articles |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2676919C (en) * | 2007-01-31 | 2013-01-29 | Allergan, Inc. | Novel biomaterials for ocular drug delivery and a method for making and using same |
| CN101418328A (en) * | 2008-10-28 | 2009-04-29 | 山东好当家海洋发展股份有限公司 | Method for producing fish scale collagen protein |
| CN101695583B (en) * | 2009-10-29 | 2012-12-26 | 中国人民解放军第三军医大学第一附属医院 | Granular biological material for tissue repair and preparation method thereof |
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- 2009-10-29 CN CN 200910191251 patent/CN101695583B/en not_active Expired - Fee Related
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- 2010-06-27 WO PCT/CN2010/074562 patent/WO2011050622A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6933326B1 (en) * | 1998-06-19 | 2005-08-23 | Lifecell Coporation | Particulate acellular tissue matrix |
| CN101400738A (en) * | 2006-03-13 | 2009-04-01 | 纳图林有限公司 | Collagen powder and collagen-based thermoplastic composition for preparing conformed articles |
| CN101195044A (en) * | 2007-12-29 | 2008-06-11 | 中国人民解放军第四军医大学 | Tissue engineered fine particle tissue and method for preparing the same |
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| WO2011050622A1 (en) | 2011-05-05 |
| CN101695583A (en) | 2010-04-21 |
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