CN101444643B - Tissue-engineered bone cooperatively modified by double genes and applications thereof - Google Patents
Tissue-engineered bone cooperatively modified by double genes and applications thereof Download PDFInfo
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- CN101444643B CN101444643B CN 200710171058 CN200710171058A CN101444643B CN 101444643 B CN101444643 B CN 101444643B CN 200710171058 CN200710171058 CN 200710171058 CN 200710171058 A CN200710171058 A CN 200710171058A CN 101444643 B CN101444643 B CN 101444643B
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Abstract
The invention relates to a tissue-engineered bone, in particular to a tissue-engineered bone cooperatively modified by double genes and applications thereof. The technical proposal of the invention is as follows: the tissue-engineered bone which is constructed by bMSCs and cooperatively modified by Nell-1 gene and BMP-2 is prepared by the following methods: (1) the Nell-1 gene and the BMP-2 gene which are mediated by adenovirus cooperatively transfect bone marrow stromal cells cultured in vitro; and (2) the products obtained by the step (1) and biological scaffold materials construct the tissue-engineered bone, thus repairing critical bone tissue defect of a lower jaw bone of a rat. The invention has the advantages that the tissue-engineered bone cooperatively modified by the Nell-1 and the BMP-2 gene repairs the critical bone tissue defect of the rat, the formed new bones in meshes of materials are more and the effect of the formation of the new bones is superior to the tissue-engineered bone separately modified by the Nell-1 or the BMP-2.
Description
Technical field
The present invention relates to a kind of tissue-engineered bone, specifically about a kind of tissue-engineered bone and application thereof of cooperatively modified by double genes.
Background technology
BMP (BMP) belongs to the transforming growth factor superfamily, wherein BMP 2,4,5,6,7 etc. and bone, cartilage, tendon, periodontal tissue, Dentinally be formed with extremely close relation, the effect of clear and definite induced osteogenesis is all arranged.But no matter the preparation of traditional bmp protein is from bone matrix, to purify, and still uses gene engineering expression, and program is complicated, yields poorly, active unstable; When reparation large tracts of land bone was damaged, the consumption of BMP was big, may react by toxigenicity; BMP implants as extrinsic protein, also possibly cause immunoreation.In recent years there is the scholar to explore the damaged reparation of application of BMP gene therapy methods accelerated bone.Wozney in 1988 etc. at first clone hBMP1-4cDNA.The clone has obtained other member in the BMP family successively subsequently, for the BMP gene therapy of bone defect repair is laid a good foundation.The carrier of BMP gene transfer respectively has characteristics, and retrovirus retrovirus can be incorporated into genes of interest in the target cell genome, but long-term expression, and no matter adenovirus target cell propagation situation all can efficiently shift, the gene transfer right and wrong of mediation are integrated; The direct transduction method of naked DNA is simple, non-immunogenicity, and safety, expense is low, and shortcoming is that transduction efficiency is low.Application of BMP gene therapy conjunctive tissue engineering promotes the formation of new bone to become present osteanagenesis hot research fields.
Nell-1 (Nel appearance I type molecule) is the new gene with osteogenic ability, and is relevant with craniosynostosis.The polypeptide of Nell-1 coding 90kDa, and finally form trimer and be secreted into outside the born of the same parents, gene order high conservative between system planted.Nearest research shows that Nell-1 promotes that the ability of skeletonization is stronger.Nell-1 osteogenic ability in certain scope is suitable with BMP-2.Ironically this gene can promote the apoptosis that osteoblastic differentiation and eventually last stage of Osteoblast Differentiation are followed; And the fibroblast in fibroblast or former generation is not acted on; Its site of action possibly be in the downstream of skeletonization signal path, perhaps promotes skeletonization through the mechanism different with the BMP ossification.
BMPs and Nell-1 promote the machine-processed different of skeletonization: BMPs is through cell-membrane receptor, and SMAD albumen, Cbf α-1 transcription factor start skeletonization; And Nell-1 thinks that at present it is in the downstream of skeletonization transcription factor Cbf α-1.Find that the collaborative marrow stromal cell skeletonization that promotes of Nell-1 and BMP-2 will help to disclose some mechanism in the skeletonization signal path.The collaborative osteogenic ability that Nell-1 and BMPs are powerful, the osseous tissue that can also be used to repairing bulk is damaged, and possibly reduce because the whole body systematicness toxic and side effects that a large amount of BMP of use cause.Using Nell-1 and the synergistically modified bMSCs compound bio of BMPs timbering material structure tissue-engineered bone has a good application prospect.
Summary of the invention
The objective of the invention is to:
(1) provide the synergistically modified bMSCs of a kind of Nell-1 and BMP-2 to make up tissue-engineered bone;
(2) the tissue-engineered bone purposes is provided.
The objective of the invention is to realize: the objective of the invention is to realize: the rat bMSCs that selects the collaborative transfection cultured and amplified in vitro of Nell-1 and BMP-2 gene through following technical method through following technical method; The Nell-1 gene is the stronger new gene of osteogenic ability; Present research is thought on cellular level; Nell-1 induces Osteoblast Differentiation to a certain degree separately; It possibly handle the downstream pathway of some skeletonization related genes, and has influenced other signal paths, promotes osteoblastic differentiation.As a kind of skeletonization related gene of new clone, Nell-1 has potential safety and tangible ossification, and its product albumen possibly become a kind of new somatomedin in the bone tissue engineer.
BMP (BMP) belongs to the TGF-beta superfamily, and wherein BMP-2,4,7 etc. all has clear and definite independent induced osteogenesis ability.Wozney at first cloned BMP-2,4 genes in 1988, thereby laid a good foundation for the BMP gene therapy of bone defect repair.The BMP gene therapy has many potential superioritys than the direct application of BMP albumen of traditional method.Can locally discharge with targeting such as, gene outcome, can increase the topical therapeutic effect to greatest extent, reduce systemic side effects, the albumen of endogenous expression possibly have bigger BA, has therefore become the focus that the bone reparation is studied.The BMP-2 gene that the present invention selects to have clear and definite induced osteogenesis effect and the synergistically modified rat bMSCs of Nell-1 make up tissue-engineered bone, the reparation of promotion jaw defect as the seed cell of tissue-engineered bone.
Marrow stromal cell (bone marrow stromal cells, it is abundant bMSCs) to have a source, it is convenient to gather, easily at In vitro culture, the characteristics of inducing, increasing, and have clear and definite skeletonization potential, be the ideal seed cell of tissue-engineered bone.This cell has the stronger multiplication capacity that goes down to posterity at In vitro culture, and the external source genes of interest is easy to import, and also is the comparatively ideal target cell of the damaged gene therapy of bone therefore.Use bMSCs and come expressing gene that many advantages are arranged, bMSCs is easy to gather and carry out tissue culture, himself has skeletonization potential.The present invention utilizes Nell-1 and BMP-2 gene to work in coordination with bMSCs such as transfection rat, Canis familiaris L., rabbit, sheep, pig, and cell and the biologic bracket material compound (like tricalcium phosphate, porous hydroxyapatite, Corallium Japonicum Kishinouye, calcium alginate, collagen etc.) with genetic modification can make up tissue-engineered bone then.The microcellular structure and the controlled bata-tricalcium phosphate of the degradation rate (β-tricalcium phosphate of the biomaterial company limited development of above sea cowry road difficult to understand; β-TCP) is an example, can play the falsework effect, the guiding osteogenesis; Bone around allowing soaks in material grows into; Material is absorbed gradually then, and NIP or rejection do not produce part or general toxic reaction.β-TCP is not homophase crystal a kind of of tricalcium phosphate.Since the seventies, β-TCP is one of focus of bone alternate material research field always from twentieth century, and its molecular formula is Ca
3(PO
4)
2, the calcium/phosphorus ratio of the synthetic β of industry-TCP powder is 1.49～1.51, and is approaching with the calcium-phosphorus ratio of normal bone tissues.Inorganic constituents [the Ca of the composition of β-TCP and bone matrix
10(PO
4)
6(OH)
2] similar, so be considered to have excellent biological compatibility.β-TCP has characteristics such as absorbing slow, low immunoreation and hypotoxicity, and has bone guided property, is suitable as the material that organizational project is repaired jawbone.It is compound as the seed cell and the timbering material of tissue-engineered bone to use the collaborative transfection bMSCs of Nell-1 and BMP-2 gene, makes up tissue-engineered bone and repairs the critical jaw defect of rat.To sum up, the present invention cultivates rat bMSCs with adherent method, with the rat bMSCs promotion bMSCs Osteoblast Differentiation of Nell-1 and the collaborative transfection In vitro culture of BMP-2 gene.
Technical scheme of the present invention is following: the synergistically modified bMSCs of a kind of Nell-1 and BMP-2 gene makes up tissue-engineered bone, and it is made by following method: the marrow stromal cell of the collaborative transfection In vitro culture of Nell-1 that (1) is adenovirus mediated and BMP-2 gene; (2) the compound structure tissue-engineered bone of product, biologic bracket material that step (1) is obtained.Adenovirus mediated Nell-1 and BMP-2 are with the marrow stromal cell of MOI=50pfu/cell transfection In vitro culture; Bone marrow substrate cell source Mus, rabbit, sheep, Canis familiaris L., Medulla Sus domestica stromal cell; Biologic bracket material is tricalcium phosphate, porous hydroxyapatite, Corallium Japonicum Kishinouye, calcium alginate, collagen; Biologic bracket material is a bata-tricalcium phosphate, and cell density is 5 * 10
7Cell suspension and the biologic bracket material of/ml are compound.
The synergistically modified bMSCs of Nell-1 and BMP-2 makes up tissue-engineered bone and in jaw defect reparation field, uses.
The tissue-engineered bone of the disclosed a kind of cooperatively modified by double genes of the present invention and application thereof; Its advantage shows: the present invention combines gene therapy and Method of Tissue Engineering; With the target cell of bMSCs as gene therapy; Use its seed cell simultaneously,, quicken skeletonization through autocrine and the paracrine action behind the synergistically modified bMSCs of gene as tissue-engineered bone.The synergistically modified tissue-engineered bone of histological examination showed Nell-1 and BMP-2 is repaired the critical osseous tissue of rat when damaged, and new bone formation effect is superior to the tissue-engineered bone that Nell-1 or BMP-2 modify separately
Description of drawings
It is long-pending big that Fig. 1: N+B organizes new surface of bone, and visible osteoblast-like cells distributes around the osseous tissue, more flaggy osteoid tissues and bone lacuna spline structure (HE dyeing, amplification 100) occur.
It is long-pending big that Fig. 2: N+B organizes new surface of bone, and visible osteoblast-like cells distributes around the osseous tissue, more flaggy osteoid tissues and bone lacuna spline structure (HE dyeing, amplification 200) occur.
Fig. 3: N organizes long-pending the formation than the N+B group of new surface of bone and lacks (HE dyeing, amplification 100).
Fig. 4: N organizes long-pending the formation than the N+B group of new surface of bone and lacks (HE dyeing, amplification 200).
Fig. 5: B organizes long-pending the formation than the N+B group of new surface of bone and lacks (HE dyeing, amplification 100).
Fig. 6: B organizes long-pending the formation than the N+B group of new surface of bone and lacks (HE dyeing, amplification 200).
The specific embodiment
Combine concrete experiment and interpretation of result thereof and corresponding Figure of description at present, promote jaw defect to repair further explain Nell-1 and the collaborative transfection marrow stromal cell of BMP-2.
The synergistically modified tissue-engineered bone of Nell-1 and BMP-2 promotes the reparation of jaw defect
One, the collaborative transfection rat bMSCs of Nell-1 and BMP-2 gene
Step 1, former generation bMSCs obtain and cultivate under the aseptic condition; Get SPF level male Fischer 344 rat tibia in 6 age in week and femur bone marrow, gained cell suspension is centrifugal, inhales and removes supernatant and the fat that floats on liquid level; Add the DMEM culture fluid; Evenly dispel, placing condition of culture is that 37 ℃ of constant temperature mists (contain 95% air, 5%CO
2), cultivate in the incubator of 100% saturated humidity.Change liquid weekly 3 times, go down to posterity when treating the cell fusion about 90%, when being cultured to for the 2nd to 3 generation, changing culture fluid is DMEM osteogenic induction culture fluid, is used for research.
When the collaborative transfection bMSCs cell of step 2, Nell-1 and BMP-2 gene grows to the 70-80% fusion, go down to posterity, cell all is attached to diapire after 24 hours; Inhaling this moment goes DMEM to induce differentiation culture liquid; Serum-free DMEM osteogenic induction culture fluid washing 2 times adds the serum-free DMEM osteogenic induction culture fluid that half amount comprises Nell-1 and each 50pfu/ cell work concentration adenovirus vector of BMP-2, behind the adenovirus vector of taking gene of adding dilution; In incubator, cultivated one hour; Every during this time even jog rolling at a distance from 15 minutes makes fully exposing cell of virus, improves transfection efficiency.The culture fluid that contains 20% serum that adds other half amount after one hour.FBS content is 10% in the culture fluid at this moment.Put incubator and cultivate after 24 hours, inhale and remove culture fluid, PBS liquid washed twice adds full dose osteogenic induction differentiation culture liquid, continues to cultivate.Changed liquid in per 3 days.Nell-1 gene and BMP-2 gene obtain from U.S. Origen company.
Two, make up tissue-engineered bone and promote the jaw defect reparation
The structure of step 1, tissue-engineered bone is with 3 days bMSCs of gene transfection digestion, and processing cell density is 5 * 10
7The cell suspension of/ml, the cylindrical beta-TCP material that slowly is added drop-wise to diameter and is 5mm make material moistening, and liquid does not flow out to saturation.
Step 2, the damaged model creation of Rat Mandibular bone and defect repair are tested will grow up under the aseptic condition Fisher 344 rat anesthesias, jaw inferior segment preserved skin, sterilization; Under the rat jaw, do the otch that is parallel to the lower jaw lower edge; Cut skin; Subcutaneous tissue and muscle-periosteal layer also turn over lobe, expose the cheek side and the levum plate of mandibular bone.Make the round defect of 5mm diameter, slowly injecting normal saline cooling all the time in the process with the drill bit of Kavo high speed machine band 5mm external diameter in mandibular ramus.In boring procedure, note protection, prevent to injure pterygoid venous plexus jawbone tongue side soft tissue.With bMSCs-β-TCP complex be filled in damaged in.Subsequently with 4-0 suture suture muscles-periosteal layer and skin-hypodermis layer.。
Step 3, draw materials, pathological study injects excessive pentobarbital with rat and causes death, sharp property separation soft or hard tissue takes out graft with bone shears, decalcification, dehydration, FFPE, section, HE dye and the skeletonization areal analysis.Compare synergistically modified tissue-engineered bone of Nell-1 and BMP-2 and the gene effect of the tissue-engineered bone skeletonization of modification separately.
Nell-1 modifies bMSCs and makes up tissue-engineered bone
Step 1, former generation bMSCs obtain and cultivate under the aseptic condition; Get SPF level male Fischer 344 rat tibia in 6 age in week and femur bone marrow, gained cell suspension is centrifugal, inhales and removes supernatant and the fat that floats on liquid level; Add the DMEM culture fluid; Evenly dispel, placing condition of culture is that 37 ℃ of constant temperature mists (contain 95% air, 5%CO
2), cultivate in the incubator of 100% saturated humidity.Change liquid weekly 3 times, go down to posterity when treating the cell fusion about 90%, when being cultured to for the 2nd to the 3rd generation, changing culture fluid is DMEM osteogenic induction culture fluid, is used for research.
When step 2, Nell-1 gene transfection bMSCs the 3rd generation cell grow to the 70-80% fusion, go down to posterity, cell all is attached to diapire after 24 hours; Inhale this moment and go DMEM to induce differentiation culture liquid, serum-free DMEM osteogenic induction culture fluid washing 2 times adds the serum-free DMEM osteogenic induction culture fluid of partly measuring working concentration Nell-1 adenovirus vector; In incubator, cultivated one hour; Every during this time even jog rolling at a distance from 15 minutes makes fully exposing cell of virus, improves transfection efficiency.The culture fluid that contains 20% serum that adds other half amount after one hour.FBS content is 10% in the culture fluid at this moment.Put incubator and cultivate after 24 hours, inhale and remove culture fluid, PBS liquid washed twice adds full dose osteogenic induction differentiation culture liquid, continues to cultivate.Changed liquid in per 3 days.The Nell-1 gene obtains from U.S. Origen company.
The structure of step 3, tissue-engineered bone is with 3 days bMSCs of gene transfection digestion, and processing cell density is 5 * 10
7The cell suspension of/ml, the cylindrical beta-TCP material that slowly is added drop-wise to diameter and is 5mm make material moistening, and liquid does not flow out to saturation.
Step 4, the damaged model creation of Rat Mandibular bone and defect repair are tested will grow up under the aseptic condition Fisher 344 rat anesthesias, jaw inferior segment preserved skin, sterilization; Under the rat jaw, do the otch that is parallel to the lower jaw lower edge; Cut skin; Subcutaneous tissue and muscle-periosteal layer also turn over lobe, expose the cheek side and the levum plate of mandibular bone.Make the round defect of 5mm diameter, slowly injecting normal saline cooling all the time in the process with the drill bit of Kavo high speed machine band 5mm external diameter in mandibular ramus.In boring procedure, note protection, prevent to injure pterygoid venous plexus jawbone tongue side soft tissue.With bMSCs-β-TCP complex be filled in damaged in.Subsequently with 4-0 suture suture muscles-periosteal layer and skin-hypodermis layer.
Step 5, draw materials, pathological study injects excessive pentobarbital with rat and causes death, sharp property separation soft or hard tissue, with bone shears taking-up graft, decalcification, dehydration, FFPE, section, the HE observation of dyeing.
BMP-2 modifies bMSCs and makes up tissue-engineered bone
Step 1, former generation bMSCs obtain and cultivate under the aseptic condition; Get SPF level male Fischer 344 rat tibia in 6 age in week and femur bone marrow, gained cell suspension is centrifugal, inhales and removes supernatant and the fat that floats on liquid level; Add the DMEM culture fluid; Evenly dispel, placing condition of culture is that 37 ℃ of constant temperature mists (contain 95% air, 5%CO
2), cultivate in the incubator of 100% saturated humidity.Change liquid weekly 3 times, go down to posterity when treating the cell fusion about 90%, when being cultured to for the 2nd to 3 generation, changing culture fluid is DMEM osteogenic induction culture fluid, is used for research.
When step 2, BMP-2 gene transfection bMSCs the 3rd generation cell grow to the 70-80% fusion, go down to posterity, cell all is attached to diapire after 24 hours; Inhaling this moment goes DMEM to induce differentiation culture liquid; Serum-free DMEM osteogenic induction culture fluid washing 2 times adds the serum-free DMEM osteogenic induction culture fluid of partly measuring working concentration BMP-2 adenovirus vector, behind the adenovirus vector of taking gene of adding dilution; In incubator, cultivated one hour; Every during this time even jog rolling at a distance from 15 minutes makes fully exposing cell of virus, improves transfection efficiency.The culture fluid that contains 20% serum that adds other half amount after one hour.FBS content is 10% in the culture fluid at this moment.Put incubator and cultivate after 24 hours, inhale and remove culture fluid, PBS liquid washed twice adds full dose osteogenic induction differentiation culture liquid, continues to cultivate.Changed liquid in per 3 days.The BMP-2 gene obtains from U.S. Origen company.
The structure of step 3, tissue-engineered bone is with the bMSCs digestion of gene transfection, and processing cell density is 5 * 10
7The cell suspension of/ml, the cylindrical beta-TCP material that slowly is added drop-wise to diameter and is 5mm make material moistening, and liquid does not flow out to saturation.
Step 4, the damaged model creation of Rat Mandibular bone and defect repair are tested will grow up under the aseptic condition Fisher 344 rat anesthesias, jaw inferior segment preserved skin, sterilization; Under the rat jaw, do the otch that is parallel to the lower jaw lower edge; Cut skin; Subcutaneous tissue and muscle-periosteal layer also turn over lobe, expose the cheek side and the levum plate of mandibular bone.Make the round defect of 5mm diameter, slowly injecting normal saline cooling all the time in the process with the drill bit of Kavo high speed machine band 5mm external diameter in mandibular ramus.In boring procedure, note protection, prevent to injure pterygoid venous plexus jawbone tongue side soft tissue.With bMSCs-β-TCP complex be filled in damaged in.Subsequently with 4-0 suture suture muscles-periosteal layer and skin-hypodermis layer.
Step 5, draw materials, pathological study injects excessive pentobarbital with rat and causes death, sharp property separation soft or hard tissue, with bone shears taking-up graft, decalcification, dehydration, FFPE, section, the HE observation of dyeing.
Experimental result
The synergistically modified tissue-engineered bone of Nell-1 and BMP-2 promotes the reparation of jaw defect
The histology HE dyeing in 8 weeks of postoperative shows that the new surface of bone of N+B group (tissue-engineered bone that Nell-1 and BMP-2 are synergistically modified) restoring area is long-pending greater than N group (Nell-1 modifies bMSCs and makes up tissue-engineered bone) and B group (BMP-2 modifies bMSCs and makes up tissue-engineered bone), and bone tissue growth does not exceed the volume range of material.It is obvious than N group and B group that N+B forms the bone effect; Visible osteoblast-like cells distributes around the osteoid tissue; More flaggy osteoid tissues and bone lacuna spline structure (Fig. 1, Fig. 2) occur, and the skeletonization of N group this moment (Fig. 3, Fig. 4) and B group (Fig. 5, Fig. 6) does not all reach the N+B group.
Claims (1)
1. the tissue-engineered bone of a cooperatively modified by double genes, it is made by following method:
(1) marrow stromal cell of the collaborative transfection In vitro culture of adenovirus mediated Nell-1 and BMP-2;
(2) the compound structure tissue-engineered bone of product, biologic bracket material that step (1) is obtained,
Wherein, Nell-1 and BMP-2 adenovirus vector working concentration are respectively the 50pfu/ cell;
Adenovirus mediated Nell-1 and BMP-2 be with the marrow stromal cell of MOI=50pfu/cell transfection In vitro culture, said bone marrow substrate cell source Mus, rabbit, sheep, Canis familiaris L., Medulla Sus domestica stromal cell,
Compound structure is meant that cell density is 5 * 10 in the step (2)
7Cell suspension and the biologic bracket material of/ml are compound, and said biologic bracket material is a bata-tricalcium phosphate.
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Non-Patent Citations (3)
| Title |
|---|
| Catherine M Cowan et al.Synergistic Effects of Nell-1 and BMP-2 on the Osteogenic Differentiation of Myoblasts.《JOURNAL OF BONE AND MINERAL RESEARCH》.2007,第22卷(第6期),第918-930页. * |
| 李建军等.人骨形态发生蛋白2腺病毒表达载体转染人骨髓基质干细胞和对其增殖及分化的影响.《中国矫形外科杂志》.2004,第12卷(第1、2期),第60-62页. * |
| 李建军等.腺病毒介导的骨形态发生蛋白2基因转染对成骨及血管化的影响.《中华显微外科杂志》.2004,第27卷(第4期),第284-285页. * |
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