CN110438073A

CN110438073A - A kind of type I collagen microcapsules of cell surface marker specific enrichment and preparation method thereof

Info

Publication number: CN110438073A
Application number: CN201910800338.8A
Authority: CN
Inventors: 徐随民
Original assignee: Nanjing Tai Sheng Biotechnology Co Ltd
Current assignee: Nanjing Tai Sheng Biotechnology Co Ltd
Priority date: 2018-09-26
Filing date: 2019-08-28
Publication date: 2019-11-12
Also published as: CN109280639A

Abstract

The invention mainly relates to type I collagen microcapsules of a kind of cell surface marker specific enrichment and preparation method thereof, the microcapsules include microballoon kernel and shell.Invention further relates to preparation method, biological efficacy detection method.The present invention also relates to the purposes that the cell microcapsules that will be prepared through the invention are used for regenerative medicine.

Description

A kind of the type I collagen microcapsules and its system of cell surface marker specific enrichment Preparation Method

Technical field

The invention mainly relates to a kind of type I collagen microcapsules of cell surface marker specific enrichment and its preparations Method, the microcapsules include microballoon kernel and shell.Invention further relates to preparation method, biological efficacy detection method.This The cell microcapsules that invention is also related to preparing through the invention are used for the purposes of regenerative medicine.

Background technique

Mescenchymal stem cell is a kind of multipotential stem cell with the of self-replication capacity and multi-lineage potential, in body It plays an important role in histoorgan stable state (Homeostasis) maintenance process after developing and reaching maturity.Its this characteristic makes It becomes the ideal seed cell of regenerative medicine, can be used for the injury repair of Various Tissues organ.

In repair process, being implanted into stem cell long-term surviving and maintaining biological characteristics is the key that guarantee its curative effect.To the greatest extent Pipe mescenchymal stem cell immunogenicity is low, and has immunoregulatory activity, but results of animal is shown, the mesenchyma of transplanting is dry Cell is not able to maintain long-term surviving.

In order to solve this problem, stem cell can be wrapped up through biomaterial, microcapsules is made.When being implanted into internal, Stem cell spatially can keep isolation with the immunocyte of body and inflammatory environment.Meanwhile oxygen, nutriment and metabolism are useless Object can effectively be exchanged by the biomaterial with semi-transparent membrane property.In this way, under the premise of not using immunosuppressor, The stem cell of implantation long-term surviving and can function.

The biomaterial is selected from natural macromolecular material, collagen, alginates, chitosan, one of hyaluronic acid Or it is a variety of.

Alginates are the salts of alginic acid.Alginic acid is a kind of poly uronic acid extracted from kelp, not soluble in water, but Its sodium salt is soluble easily in water.Alginate soln passes through and bivalent cation, such as Ca²⁺Crosslinking can form gel.Alginate jelly is nontoxic, It will not cause to be immunoreacted after being injected in vivo, but the disadvantage is that hole is greatly and unstable.

Chitosan is the derivative of chitin, covalent cross-linking can occur with Geniposide, have nontoxic, bio-compatible after crosslinking The features such as property is good, anti-inflammatory.

One layer of chitosan film is wrapped up outside alginate jelly, the chitosan film of covalent cross-linking can provide better stability. Stable microcapsules can extend the residence time of cell in vivo, improve its validity.

The size of microcapsules has larger impact to Cell viability therein is wrapped up.The diameter of microcapsules is too big (> 500 μm), The cellular invasion at nutrients and oxygen to microcapsules center is insufficient, and metabolic waste cannot be discharged in time, will lead under Cell viability Drop.Micro-fluidic technologies can prepare that particle diameter distribution is uniform, and microcapsules of the diameter less than 500 μm.

Dental pulp stem cell is one kind of mescenchymal stem cell, and passes through the subgroup of cell surface marker specific enrichment, excellent Select CD18⁺Dental pulp stem cell subgroup has stronger clonality, more preferably Osteoblast Differentiation ability, have superior scientific research and Potential applicability in clinical practice.

Summary of the invention

The present invention provides a kind of type I collagen microcapsules of cell surface marker specific enrichment and its preparation sides Method, the microcapsules include microballoon kernel and shell.Specifically, being achieved by the following technical solution:

This method, which is used containing there are many combinations of digestive ferment, digests adult pulp tissue, obtains individual cells suspension.Including But be not limited to clostridiopetidase A, dispase, hyaluronidase, pancreatin, EDTA etc., the combination of these digestive ferments is greatly improved obtaining for cell Rate.

The use concentration of clostridiopetidase A can be 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ in this method Ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.The use concentration of dispase can be 1 mg/ml, and 2 Mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.

This method is by cell surface marker specific enrichment, and preferably CD18 is enriched with dental pulp stem cell subgroup, CD18 Fluorescent marker or marked by magnetic bead can be used.Correspondingly, accurate cell separation technology includes selected by flow cytometry apoptosis, or exempt from Epidemic disease magnetic bead sorting.

The amplification in vitro basal medium of CD18 Positive Stem Cells can be α-MEM, DMEM, D/F-12 in this method, RPMI-1640 etc..Complete medium is to add fetal calf serum, ascorbic acid 2- phosphate, paddy on the basis of basal medium Glutamine, penicillin, streptomysin etc..

The concentration of fetal calf serum can be 5%, 10%, 15%, 20% in complete medium.The concentration of ascorbic acid 2- phosphate It can be 50 μM, 100 μM, 150 μM, 200 μM.Other additive concentration are added according to art-recognized prioritization scheme.

The amplification passage ratio of CD18 Positive Stem Cells can be 1:3,1:4 or 1:5 in this method.It is used to prepare micro- glue The cell of capsule was the 4th generation, the 5th generation or the 6th generation.

The alginate soln concentration that cell microsphere is used to prepare in this method can be 1%, 2% or 3%.Prepared solution It need to be through 0.22 μm of membrane filtration degerming.

It is 1 × 10 that the concentration of cell, which is resuspended, in alginate soln in this method⁶/ ml, 1.5 × 10⁶/ ml, or 2 × 10⁶/ml。

The microfluidic device that cell microsphere size is controlled in this method is horizontal, vertical binary channels design, containing cell Alginate soln is instilled straight down in the plate equipped with calcium chloride solution, forms cell microsphere.The flowing of soybean oil horizontal cross, Alginate soln is truncated, controls the size of cell microsphere.The cell microsphere diameter prepared by the microfluidic device is less than 500 μ m。

Calcium chloride solution concentration used in cell microsphere is prepared in this method can be 0.1M or 0.2M, prepared molten Liquid need to be through 0.22 μm of membrane filtration degerming.

It need to be incubated for 15 minutes, 20 points in calcium chloride solution under the conditions of 37 DEG C by cell microsphere prepared by this method Clock, 25 minutes, 30 minutes, 35 minutes, 40 minutes or 45 minutes.

The chitosan solution concentration that cell microcapsules are used to prepare in this method can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.Prepared solution need to be through 0.22 μm of membrane filtration degerming.

It is 15 minutes with time of chitosan solution coated cell microballoon, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 Minute, 45 minutes.

It can be 1mg/ml, 2mg/ml, 3mg/ml for the concentration with chitosan crosslinked genipin solution in this method, 4mg/ml, 5mg/ml.Prepared solution need to be through 0.22 μm of membrane filtration degerming.

The condition of chitosan and genipin cross-linked is incubated for 1 hour under the conditions of being 37 DEG C in this method, and 2 hours, 3 hours, 4 is small When, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours.

The present invention also provides a kind of detection methods of cell microcapsules exobiology effect.Specifically, being to pass through What following technical solution was realized:

This method is using the external Osteoinductive differentiation ability of cell microcapsules as the side of assessment cell microcapsules biological efficacy Method.

Osteogenic Induction Medium used is to add dexamethasone, vitamin C and β-phosphorus on the basis of basal medium Acid glycerol etc..

The use concentration of dexamethasone can be 0.05mM, 0.1mM, 0.15mM, 0.2mM in Osteogenic Induction Medium, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM.

It is ascorbic to can be 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/ using concentration ML, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL.

β-phosphoglycerol use concentration can be 5 mM, 6 mM, 7 mM, 8mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM.

The cell microcapsules of external evoked culture 4 weeks are made histotomy by this method, are detected by Alizarin red staining The Osteoblast Differentiation ability of cell in microcapsules, to assess the biological efficacy of cell microcapsules.

Studies have shown that Runx2 (runt-related transcription factor 2) is important during bone development Transcription factor, can be used as Osteoblast Differentiation detection marker molecule.This method detects Runx2 mRNA table by RT-PCR method Up to level, Western Blots method detects Runx2 protein translation level, and the skeletonization for further analyzing cell in microcapsules lures Lead differentiation capability.

Cell microcapsules prepared by the present invention can be directly used for regenerative medicine.

Detailed description of the invention

Fig. 1 CD18 Positive Stem Cells subgroup

Fig. 2 CD18 Positive Stem Cells subgroup phenotypic analysis

Fig. 3 microcapsules Preparation equipment

Fig. 4 CD18 Positive Stem Cells microcapsules

The analysis of Fig. 5 differentiation capability

The detection of Fig. 6 Runx2 mRNA expression

The detection of Fig. 7 Runx2 protein level.

Specific embodiment

The separation and culture of 1 CD18 positive dental pulp stem cell of embodiment

Materials and methods

1. collecting normal people (19-29 years old) extraction of wisdom tooth, it is immediately placed on penicillin containing 50-300U/mL, the 50- of 4 DEG C of pre-coolings In the α-MEM culture medium of 300 μ g/mL streptomysins and 1-5 μ g/mL amphomoronal, clean experiment is transported under the conditions of 2 DEG C -8 DEG C Room.Since the time interval that dental pulp stem cell is extracted being collected into be no more than 72 hours；

2. being transferred to Biohazard Safety Equipment, wisdom tooth is taken out to culture dish, with the sterile phosphate buffer (Phosphate of 4 DEG C of pre-coolings Buffer Saline, PBS) clean its outer surface；

3. being cut with sterile fissure bur from cementum and enamel intersection, exposure pulp chamber gently separates pulp tissue；

4. pulp tissue is placed in the PBS of the collagenase type I of mg/ml containing 1-10 and 1-10 mg/ml dispase, 37 DEG C of digestion 1 Hour；

5. digestive juice is filtered by 70 microns of cell sieve, single cell suspension is obtained；

6.900rpm is centrifuged 5min, and it is primary to wash cell with PBS；

7. sorting by fluidic cell, CD18 Positive Stem Cells subgroup is obtained；

8. by the positive cell of sorting be inoculated in fetal calf serum containing 10%-20%, 50 μM -200 μM of ascorbic acid 2- phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium in, be placed in 37 DEG C, 5% CO₂Incubate It is cultivated in case；

9. every 3-4 days change liquid, after the fusion of cell 90%, 1:3 passage, preparing cell used in microballoon is forth generation.

The results show that the cell of culture is spindle, in circinate growth (Fig. 1), cell phenotype CD18 is positive (Fig. 2).

The preparation of 2 cell microcapsules of embodiment

Materials and methods

1. alginates are dissolved in PBS, it is made into the alginate soln of 1-3%, through 0.22 μm of membrane filtration, packing is saved；

2. the CD18 positive dental pulp stem cell of culture to forth generation is digested through pancreatin, 900rpm is centrifuged 5min, is washed carefully with PBS Born of the same parents are primary；

3. the CD18 positive dental pulp stem cell of harvest is resuspended with alginate soln, adjustment cell concentration is 1-2 × 10⁶/ml；

4. by horizontal, vertical binary channels microfluidic device as shown in Figure 3, by alginate soln containing cell through channel 1 It instills in the plate equipped with 0.1-0.2M calcium chloride solution straight down, forms cell microsphere.Soybean oil is through 2 horizontal cross of channel Flowing is truncated alginate soln, controls the size of cell microsphere；

5. microballoon is incubated for 15-45 minutes under the conditions of 37 DEG C, to fully crosslinked formation；

6. alginates cell microsphere is coated with 15-45 minutes in 0.1-1% chitosan solution；

It is incubated for the crosslinking of completion in 1-10 hours 7. being subsequently placed in 1-5 mg/ml Geniposide aqueous solution under the conditions of 37 DEG C, prepares cell Microcapsules.

8. cell microcapsules are cleaned and collected with α-MEM, and it is inoculated in fetal calf serum containing 10%-20%, 50 μ α-MEM the culture of M-200 μM of ascorbic acid 2- phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins In base, it is placed in 37 DEG C, 5% CO₂Incubator in cultivate；

9. using the shape and diameter of the cell microcapsules that optical microphotograph sem observation is formed.

The results show that the cell microcapsules of preparation are in spherical, it is uniform in size.Cell is uniformly distributed wherein, rounded (figure 4).

The detection of 3 cell microcapsules exobiology effect of embodiment

Materials and methods

1. collecting cell microcapsules, and it is inoculated in the ascorbic acid 2- phosphoric acid of fetal calf serum containing 10%-20%, 50 μM -200 μM Ester, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium in, be placed in 37 DEG C, 5% CO₂'s It is cultivated in incubator；

2. after culture 24-48 hours, being replaced with Osteogenic Induction Medium (containing 10% fetal calf serum, 100U/mL penicillin, 100 μ G/mL streptomysin, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β-phosphoglycerol α-MEM culture medium)；

3. every 3-4 days change liquid；

4. after Fiber differentiation 4 weeks, fixing cell microcapsules 30 minutes under room temperature with 4% paraformaldehyde；

5. setting in PBS 15 minutes；

6. dehydration, transparent, paraffin embedding are sliced, dewaxing, rehydration；

7. cell microcapsules histotomy is placed in 2% alizarin red aqueous solution, room temperature dyes 5min；

8. after rinsing with ruinning water, using haematoxylin redyeing；

9. mounting microscopy.

The results show that passing through Alizarin red staining (Fig. 5), it is seen that in microcapsules in vitro after osteogenic induction culture in 3-4 weeks Iuntercellular has mineral deposition, shows CD18 in microcapsules⁺Dental pulp stem cell can not be influenced to osteoblast differentiation, encapsulation Its biological characteristics.

The detection of 4 cell Runx2 mRNA expression of embodiment

Materials and methods

1.5×10⁵The Trizol(Invitrogen company of 1ml is added in a dental pulp stem cell) cracking；

2. 200 μ L chloroforms are added, acutely concussion 15 seconds, mix, be placed at room temperature for 3min；

3.4 DEG C, 12000rpm/min, it is centrifuged 15min；

4. careful Aspirate supernatant is transferred in the EP pipe of new no RNA enzyme, isometric isopropanol is added, sufficiently mixed It is even, it is placed at room temperature for 10min；

5.4 DEG C, 12000rpm/min, it is centrifuged 10min；

6. abandoning supernatant, the ethyl alcohol of 500 μ L 75% of pre-cooling is added, turns upside down for several times, it is seen that precipitating；

7.4 DEG C, 12000rpm/min, it is centrifuged 10min；

Supernatant is abandoned 8. inhaling, is dried, appropriate DEPC water dissolution RNA is added, surveys OD value.

9. the total serum IgE extracted in right amount is taken to carry out reverse transcription.Illustrate to prepare according to Reverse Transcriptase kit (Takara company) The reaction system of 20 μ L, response procedures are as follows:

37 DEG C, 15min × 3

85 DEG C, 5sec

After the completion of reverse transcription reaction, the cDNA of acquisition is frozen and is saved in -80 DEG C of refrigerators.

10. utilizing Takara company using cDNA as templateTaqPolymerase carries out fluorescence real-time quantitative PCR, detects Runx2 MRNA(primer: forward, 5 '-CAGTTCCCAAGCATTTCATCC-3 '; reverse, 5‘- TCAATATGGTCGCCAAACAG-3 ') expression, GAPDH as reference gene (primer: forward, 5 '- AGCCGCATCTTCTTTTGCGTC-3';Reverse, 5 '-TCATATTTGGCAGGTTTTTCT-3 ').

11. reaction system is as follows:

SYBR Premix Ex Taq II(2X) 12.5 μ L

PCR Forward Primer(10 μM) 1 μ L

PCR Reverse Primer(10 μM) 1 μ L

2 μ L of cDNA solution

ddH2O 8.5μL

After above-mentioned 25 μ L reaction mixture of system configurations, it is added in eight unions, and set 3 multiple holes.Reaction condition are as follows:

Step 1:95℃ 30sec

Step 2:PCR reaction

GOTO:40 circulation

95℃ 5sec

60℃ 30sec

Step 3:Melt Curve

12. PCR sample is taken to carry out agarose gel electrophoresis, data analysis.

The results show that inducing Runx2 mRNA expression in noble cells significant with differentiation control cell ratio is not induced Higher than cellular control unit (Fig. 6).

The detection of 5 cell Runx2 protein level of embodiment

1. the protein lysate (Suo Laibao company) of pre-cooling is added in dental pulp stem cell, it is placed in and cracks 30min on ice；

2.4 DEG C, 12000rpm/min, it is centrifuged 15min；

3. the supernatant after careful transfer centrifugation freezes spare in -80 °C of refrigerators into new Ep pipe；

4. detecting total protein concentration using BCA protein quantification kit (green skies company)；

5. protein lysate and 5 × loading buffer are added in the sample containing 20 μ g albumen, mixing is placed on 95 ° 5min makes its denaturation in C water-bath；

6. carrying out SDS-PAGE electrophoresis using 12% separation gel and 4% concentration glue；

7. after electrophoresis, glue is cut, and be close to an equal amount of nitrocellulose filter (PVDF), in the transferring film buffer of pre-cooling Middle transferring film；

8. after, PVDF film is taken out, is placed in 10% balf serum albumin of film washing liquid preparation, room temperature closing 1h；

9. using antibody incubation liquid dilution primary antibody (anti-Runx2, anti-β-actin, Abcam), 4 °C of incubations are placed in shaking table On rock overnight；

10. next day removes primary antibody, film washing liquid is cleaned 3 times, every all over 10min；

11. being incubated for secondary antibody 1h at room temperature；

12. PVDF film is placed in Labworks image acquisition and analysis software, immunoblotting chemiluminescence in drop after film washing liquid cleans three times Liquid makes the surface of the uniform whole cover films of luminescent solution, after being incubated for 1min, scans appropriate time, record saves image.

13. interpretation of result.

The results show that and do not induce differentiation control cell ratio, induce Runx2 protein level in noble cells to be significantly higher than pair According to a group cell (Fig. 7).

Claims

1. a kind of type I collagen microcapsules by cell surface marker specific enrichment, it is characterised in that:

The stem cell is type I collagen, and the man-year age is 19 to 29 years old, and the cell surface marker is selected from CD18, STRO-1, CD106/VCAM-1, CD146/MUC-18, HOP-26, CD49A/ integrin β_1, one in SB-10/CD166 or It is multiple；

The microcapsules include microballoon kernel and shell, and the microsphere diameter is less than 500 μm；

The microencapsulation material is selected from collagen, alginates, chitosan or hyaluronic acid；

The microcapsules detect Runx2 mRNA expression by RT-PCR method, and Western Blots method detects Runx2 Protein translation is horizontal.

2. type I collagen microcapsules described in claim 1, which is characterized in that using CD18 as cell surface marker, institute It states CD18 and uses fluorescent marker or marked by magnetic bead.

3. type I collagen microcapsules as claimed in claim 2, which is characterized in that the microcapsules inner nuclear material is selected from alginic acid Salt；Sheathing material is selected from chitosan film, and the chitosan film is covalent cross-linking.

4. the preparation method of type I collagen microcapsules described in a kind of claim 3, which is characterized in that the method it is specific Steps are as follows:

Step 1, alginates are dissolved in PBS, are made into the alginate soln of 1-3%, through 0.22 μm of membrane filtration, packing is protected It deposits；

Step 2, the CD18 positive dental pulp stem cell by culture to forth generation is digested through pancreatin, and 900rpm is centrifuged 5min, is washed with PBS Cell is primary；

Step 3, be resuspended the obtained CD18 positive dental pulp stem cell of step 2 with alginate soln, adjustment cell concentration be (1-2) × 10⁶/ml；

Step 4, by horizontal, vertical binary channels microfluidic device, alginate soln containing cell is dripped downward into through channel In plate equipped with 0.1-0.2M calcium chloride solution, cell microsphere is formed；It is molten that alginates are truncated through channel cross-flow in soybean oil Liquid controls the size of cell microsphere；

Step 5, microballoon is incubated for 15-45 minutes under the conditions of 37 DEG C, to fully crosslinked formation；

Step 6, alginates cell microsphere is coated with 15-45 minutes in (0.1-1) % chitosan solution；

Step 7, it is subsequently placed in 1-5 mg/ml Geniposide aqueous solution under the conditions of 37 DEG C and is incubated for the crosslinking of completion in 1-10 hours, preparation Cell microcapsules；

Step 8, cell microcapsules are cleaned and are collected with α-MEM, and be inoculated in fetal calf serum containing 10%-20%, 50 μM- 200 μM of ascorbic acid 2- phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium In, it is placed in 37 DEG C, 5% CO₂Incubator in cultivate；

Step 9, the shape and diameter of the cell microcapsules formed using optical microphotograph sem observation.

5. the preparation method of type I collagen microcapsules described in a kind of claim 4, which is characterized in that the culture to the 4th The preparation step of the CD18 positive dental pulp stem cell in generation is as follows:

Step 1, normal people's extraction of wisdom tooth is collected, penicillin containing 50-300U/mL, the 50-300 μ g/ of 4 DEG C of pre-coolings are immediately placed on In the α-MEM culture medium of mL streptomysin and 1-5 μ g/mL amphomoronal, it is transported to Clean Operating Lab under the conditions of 2 DEG C -8 DEG C, from receipts Collect the time interval for starting to extract dental pulp stem cell to be no more than 72 hours, the normal man-year age was at 19 to 29 years old；

Step 2, it is transferred to Biohazard Safety Equipment, takes out wisdom tooth to culture dish, the sterile phosphate buffer being pre-chilled with 4 DEG C cleans, That is PBS cleans its outer surface；

Step 3, it is cut with sterile fissure bur from cementum and enamel intersection, exposure pulp chamber gently separates pulp tissue；

Step 4, pulp tissue is placed in the PBS of the collagenase type I of mg/ml containing 1-10 and 1-10 mg/ml dispase, 37 DEG C Digestion 1 hour；

Step 5, digestive juice is filtered by 70 microns of cell sieve, obtains the single cell suspension containing type I collagen, 900rpm is centrifuged 5min, and it is primary to wash cell with PBS；

Step 6, cell is resuspended with PBS, CD18 monoclonal antibody is added, mix, room temperature is protected from light incubation 30 minutes；

Step 7, after washing, 900rpm is centrifuged 5min, and cell is resuspended with PBS；

Step 8, by selected by flow cytometry apoptosis or immunological magnetic bead sorting, CD18 Positive Stem Cells subgroup is obtained；

Step 9, liquid is changed within every 3-4 days, after the fusion of cell 90%, 1:3 passage, preparing cell used in microballoon is forth generation.

6. a kind of stemness detection method of type I collagen microcapsules described in claim 1-3, which is characterized in that the method Specific step is as follows:

Step 1, cell microcapsules are collected, and are inoculated in the ascorbic acid 2- phosphorus of fetal calf serum containing 10%-20%, 50 μM -200 μM Acid esters, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium in, be placed in 37 DEG C, 5% CO₂ Incubator in cultivate；

Step 2, after cultivating 24-48 hours, it is replaced with Osteogenic Induction Medium, that is, contains 10% fetal calf serum, 100U/mL mould Element, 100 μ g/mL streptomysins, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β-phosphoric acid α-MEM the culture medium of glycerol, changes liquid in every 3-4 days；

Step 3, after Fiber differentiation 4 weeks, cell microcapsules 30 minutes are fixed under room temperature with 4% paraformaldehyde；

Step 4, it sets in PBS 15 minutes；

Step 5, it is dehydrated, transparent, paraffin embedding, is sliced, dewaxing, rehydration；

Step 6, cell microcapsules histotomy is placed in 2% alizarin red aqueous solution, room temperature dyes 5min；

Step 7, after rinsing with ruinning water, haematoxylin redyeing is used；

Step 8, mounting microscopy.

7. a kind of purposes of type I collagen microcapsules described in claim 1-3, which is characterized in that the regeneration for bone disease The preparation of medicine and tissue engineering material.

8. a kind of purposes of type I collagen microcapsules described in claim 1-3, which is characterized in that will by transgenic technology CD18 gene is transferred to stroma stem cell, the preparation of regenerative medicine and tissue engineering material for bone disease.

9. the purposes of type I collagen microcapsules described in claim 8, which is characterized in that the stromal stem cell source choosing From tooth, fat, marrow, umbilical cord, placenta or bleeding of the umbilicus.

10. the purposes of type I collagen microcapsules described in claim 8, which is characterized in that the transgenic technology is to pass through The mode of retrovirus-mediated method.