WO2014190591A1 - 一种组织工程关节双相支架及其制备方法和应用 - Google Patents
一种组织工程关节双相支架及其制备方法和应用 Download PDFInfo
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- 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
- A61L27/38—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 containing added animal cells
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- 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
- A61L27/26—Mixtures of macromolecular compounds
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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
- A61L27/36—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
- A61L27/38—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 containing added animal cells
- A61L27/3839—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 containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3847—Bones
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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
- A61L27/36—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
- A61L27/38—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 containing added animal cells
- A61L27/3839—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 containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
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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
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
Definitions
- the present invention relates to the field of tissue engineering technology, and more particularly to a tissue engineering joint biphasic stent and a preparation method and application thereof.
- This biphasic scaffold consists of a bone-phase scaffold and a cartilage-phase scaffold for the construction of bone and joint soft bones, respectively, and the two are perfectly matched.
- tissue engineering technology provides a possibility for the regeneration of tissue-engineered joints.
- seed cells are inoculated on appropriate joint scaffolds, supplemented by cytokines to regenerate tissue-engineered joints in vivo. This is a true sense.
- the bio-type joints provide a possibility for tissue-engineered joints to replace the existing artificial joints for bioreconstruction of joints.
- the size and shape of the tissue-engineered joint should be precisely matched to the normal joint.
- the interface between the regenerated joint, including the cartilage and the subchondral bone, is similar or identical to the normal tissue structure.
- the regenerated tissue has the biological and mechanical biomechanical properties of normal tissue. Wait. But so far, there has been no breakthrough in the regeneration of tissue engineered joints. Summary of the invention
- the object of the present invention is to provide a tissue engineering joint biphasic stent for the deficiencies in the prior art.
- a second object of the present invention is to provide a method of preparing a tissue engineered joint biphasic scaffold.
- a third object of the present invention is to provide an application of a tissue engineered joint biphasic stent.
- a fourth object of the present invention is to provide a cell-tissue engineering joint biphasic scaffold construct.
- a fifth object of the present invention is to provide a method of preparing a cell-tissue engineering joint biphasic scaffold construct.
- a sixth object of the invention is to provide a cell-tissue engineered joint biphasic scaffold construct.
- the technical solution adopted by the present invention is: a tissue engineering joint biphasic stent, wherein the biphasic stent is composed of a bone phase scaffold and a cartilage phase scaffold, and the bone phase scaffold is polylactic acid/polyglycolic acid. (PGA/PLA) Stent, the cartilage phase scaffold is a poly- ⁇ -caprolactone/hydroxyapatite (PCL/HA) scaffold.
- the method for preparing a tissue engineering joint biphasic stent is characterized in that it comprises the following steps: first laser scanning or CT and MRI scanning, and then computer aided design
- the bone phase scaffold and the soft bone scaffold constitute a tissue engineering joint biphasic scaffold, wherein the bone phase scaffold is a PCL/HA scaffold and the cartilage phase scaffold is a PGA/PLA scaffold.
- the technical solution adopted by the present invention is: the application of the tissue engineering joint biphasic scaffold in the preparation of tissue engineering joints.
- the technical solution adopted by the present invention is: a cell-tissue joint biphasic scaffold construct, the cell-joint biphasic scaffold construct consisting of an osteogenesis portion and a cartilage portion,
- the osteogenic part is a bone marrow stromal stem cell-PCL/HA bone phase scaffold
- the chondrogenic part is a chondrocyte or bone marrow stromal stem cell-PGA/PLA cartilage phase scaffold.
- the preparation method of the osteogenic part The PCL/HA bone phase scaffold is prepared by the method of claim 2, and the bone marrow stromal stem cells are inoculated in the scaffold, and then subjected to in vitro osteogenic induction culture for 2-6 weeks.
- the preparation method of the chondrogenic part The PGA/PLA cartilage phase scaffold is prepared by the method of claim 2, and the chondrocyte or bone marrow stromal stem cells are inoculated in the scaffold; and then cultured in vitro for 2-6 weeks.
- the technical solution adopted by the present invention is: the method for preparing the cell-tissue engineering joint biphasic scaffold construct, comprising the following steps: (1) using the method of claim 4 The osteogenesis was prepared by inoculating bone marrow stromal cells in a PCL/HA bone phase scaffold, followed by in vitro osteogenic induction for 2-6 weeks. (2) A cartilage portion is prepared by the method of claim 4, and a chondrocyte or bone marrow stromal stem cell is seeded in a PGA/PLA cartilage phase scaffold; and then cultured in vitro for 2-6 weeks.
- the technical solution adopted by the present invention is: the use of the cell-tissue engineering joint biphasic scaffold construct in the preparation of tissue engineered joints.
- the tissue engineering joint biphasic scaffold of the invention is processed on the basis of computer aided design/computer aided manufacturing (CAD/CAM) technology, the external shape and internal structure are precisely controlled, and the bone phase and the cartilage phase scaffold are matched, and then Using tissue engineering techniques, composite seed cells form a cell-tissue joint biphasic scaffold construct that regenerates tissue-engineered joints in vivo. Regenerated articular cartilage and subchondral bone have the biological and mechanical biomechanical properties of normal tissues. And the interface between the two is well integrated, which provides a possibility for the future of tissue engineering joints to replace the existing artificial joints for bioreconstruction of joints.
- CAD/CAM computer aided design/computer aided manufacturing
- Figure 1 Flow chart of tissue engineering femoral head regeneration experiment
- Isolation, culture, inoculation of goat articular chondrocytes onto PGA/PLA scaffolds and in vitro culture A-B1-C1-D1
- Isolation, culture of bone marrow stromal stem cells, inoculation onto PCL/HA scaffolds and induction culture in vitro A -B2-C2-D2
- the above two cell-scaffold complexes constitute a femoral head graft (E); the above graft was implanted into the back of a nude mouse for 10 weeks (F).
- the goat femoral head laser was scanned and the PCL/HA bone phase scaffold (G-H-I-J) was processed by CAD/CAM technology.
- the PGA/PLA cartilage phase scaffold was pressed by the mold prepared by CAD/CAM technology.
- Part A Surface morphology of PGA/PLA and PCL/HA and biocompatibility of materials
- PGA/PLA stent (A1); scanning electron microscope ( ⁇ 200), the arrow shows the coating of PLA on the PGA fiber and the connection formed by it, which helps to maintain the shape of the stent (A2); the chondrocytes under the electron microscope Adhesion and stretching on the surface and formation of extracellular matrix (A3).
- PCL/HA stent (A4); scanning electron microscopy (> ⁇ 35) (A5); electron microscopy ( ⁇ 500) adhesion and extension of bone marrow stromal stem cells on the stent (A6).
- Part B Effects of different contents of PLA and HA on the characteristics of PGA/PLA and PCL/HA stents
- Part C Range of elastic modulus of PGA/PLA and PCL/HA stents with normal cartilage and cancellous bone.
- Bone marrow mesenchymal stem cells are a class of adult stem cells with multipotential differentiation potential.
- the surrounding environment includes the elastic modulus or hardness of the stent.
- the elastic modulus of PCL/HA stent is within the range of normal cancellous bone elastic modulus, which helps to further improve the ability to differentiate into osteogenesis.
- the elastic modulus of PGA/PLA stent ranges from the elastic modulus of normal articular cartilage. Within the scope, this helps to further improve the ability to differentiate into cartilage.
- FIG. 3 Construction of tissue-specific dual-phase scaffolds based on CAD/CAM technology
- the PGA/PLA stent obtains the cartilage phase data by computer Boolean subtraction operation, and then prepares the cartilage phase stent mold through the above-mentioned CAD/CAM technology three-dimensional printing processing, and prepares the soft mold by mold processing.
- Bone phase PGA/PLA stent F, 1). Constructed a femoral head biphasic stent (G).
- Figure 4 Cell-scaffold composites under optical microscopy, naked eye and in vivo implants and specimens.
- Articular chondrocytes-PGA/PLA scaffold under the light microscope the arrow shows the adhesion and growth on the scaffold after 2 weeks of cell inoculation (A); BMSCs-PCL/HA scaffold under the light microscope, the arrow shows 2 weeks after inoculation Adhesion and growth of BMSCs in microtubules of PLC/HA stents (B); these two cell-scaffold complexes combine to form a femoral head graft (C); nude mice dorsal subcutaneous graft (arrow Show) (D); 10 weeks later, the cartilage-like cartilage-like tissue was regenerated on the joint surface of the femoral head graft, and white hard bone-like tissue was grown in the tiny tubing of the PLC/HA stent, (E); The cartilage tissue regenerated throughout the articular surface of the bone is smooth, continuous, non-vascularized and well integrated with the subchondral bone tissue without visible fissures and stratification (F).
- FIG. 5 Regenerated cartilage tissue and cartilage and subchondral bone interface
- Cartilage (A) The tissue of the femoral head was smooth and continuous throughout the articular surface. No revascularization was found in the regenerated cartilage tissue, and chondrocytes were found in the extracellular matrix lacuna. The tissue was positive for Safranin 0, suggesting that cartilage tissue occurred in the articular surface of the femoral head in the experimental group (Fig. 5A, Sahong 0/fast green), indicating that the regenerated cartilage tissue has the same function of secreting aggrecan as normal cartilage tissue. Type II collagen is immunolocalized to the extracellular matrix of chondrocytes (Fig. 5A Type II collagen). In addition, most PGA/PLA scaffold materials have been degraded and only a very small amount of material remains (arrows).
- regenerated cancellous bone tissue Fig. 6 ⁇ ⁇ 0 0/ ⁇ ⁇ 40.
- the regenerated cancellous bone has obvious bone marrow tissue (Fig. 6 ⁇ Goldner tricolor ⁇ 40). It can be seen that a typical bone cell (arrow) is a common cell with 1-2 nucleoli (Fig. 6 ⁇ toluidine blue ⁇ 40).
- the regenerated bone tissue contains red blood cells (arrows) in the lumen composed of the vascular endothelium (Fig. 6 ⁇ hematoxylin-eosin 40). Bone marrow-like tissue under high power optical microscope (Fig. 6 ⁇ Goldner tricolor X 40).
- Polymethyl methacrylate embedded hard tissue plate staining (B): von Kossa staining showed in PLC/HA scaffolds There are positive calcification deposits in the connected three-dimensional microchannels (Fig. 6B von Kossax lO); toluidine blue staining shows excellent bone tissue growth and has a trabecular structure, and the arrows indicate the pores formed by degradation of the PCL/HA scaffold material ( Figure 6B Toluidine blue oxime 10).
- Osteopontin is positive in the osteogenic part. Osteopontin is an extracellular matrix cell adhesion protein that can be biosynthesized not only from preosteoblasts, osteoblasts, bone cells, but also from vascular endothelial cells. The arrow shows the regenerated vascular tissue ( ⁇ ). Type I collagen ( ⁇ ), osteocalcin (C) and osteonectin (D) were also positively expressed, respectively, and they were consistent with the distribution of bone tissue and the von Kossa positive distribution. Ruler: 200 ⁇
- Regenerated cartilage fraction We performed cartilage partial scoring according to the visual histology scoring criteria of the International Cartilage Rehabilitation Committee Tissue Society (Fig. 8 ⁇ ), and measured the number of chondrocytes/total cartilage area (Fig. 8 ⁇ ). There was no significant difference between the above quantitative analysis experiment group and the normal control group. However, there were significant differences between the experimental group and the control group.
- Regenerated bone fraction We measured bone volume/total tissue volume (BV/TV) (Fig. 8C), bone surface area/bone volume (BS/BV) (Fig. 8D), total osteoblast surface/total tissue area (Fig. 8D) Ob.S/T.Ar) (Fig. 8E) and total number of blood vessels/total tissue area (Fig. 8F). All of the above measurements showed no significant difference between the experimental group and the normal control group, but there was a significant difference between the experimental group and the control group.
- the PGA/PLA scaffolds described herein refer to PGA and PLA composite scaffolds
- the PCL/HA scaffolds refer to PCL and HA composite scaffolds, that is, "/" means that the front and the back are “and” rather than " Or "the relationship.
- the compression modulus of the polyglycolic acid/polylactic acid (PGA/PLA) scaffold increased gradually, but the chondrocyte inoculation efficiency decreased gradually.
- the PGA/PLA cartilage phase scaffold with a 10% PLA ratio fully balances mechanical properties and cell seeding efficiency.
- PCL/HA Poly- ⁇ -caprolactone/hydroxyapatite
- the PCL/HA stent has good cell adhesion.
- the PCL/HA stent has a steeper linear elastic region and a plateau region with a higher compressive force, and the compression modulus and compressive force are correspondingly increased.
- the water absorption rate also increases accordingly.
- the HA content is more than 40%, the PCL/HA fuse cannot be sprayed when the stent is formed by rapid prototyping, so we choose a PCL/HA stent with a 40% HA ratio.
- a 10-month-old goat is used as a seed cell: a source of cartilage and bone marrow stromal stem cells.
- a small amount of articular cartilage tissue was taken from the intercondylar fossa of the goat's knee joint, shredded, washed with PBS CHyClone, Logan, UT, USA), then 0.1% collagenase (Worthington Inc., Lakewood, NJ, USA) was shaken at 37 °C. The bed was incubated for 8-12 hours, and finally filtered through a 100 ⁇ filter and inoculated into a Petri dish at 5% C0 2 at 37 °C.
- bone marrow extracted from a goat knee proximal tibia condyle, isolated and inoculated into culture dishes, culturing the cell seeding density of monocytes 5 ⁇ 10 5 / cm 2, 5% C0 2 , 37 ° C conditions.
- the culture was continued using the osteogenic induction fluid from the P2 generation. It contained 10 mM ⁇ -glycerophosphate disodium (Sigma-Aldrich, St. Louis, MO, USA), ⁇ . ⁇ dexamethasone (Sigma-Aldrich) and 50 ⁇ 2-phosphate ascorbic acid (Gibco).
- the bone-based stent includes a femoral head and a shank inserted into the medullary cavity for surgical insertion and fixation in the medullary cavity, the size is 18x l7x l5mm 3 (length X width X height), and normal goat femoral head The size and shape are the same. Since this size far exceeds the distance of 200 ⁇ m of the material exchange of the regenerated tissue, three-dimensional micro-pipes (200-400 ⁇ m in diameter) interconnected inside each other are designed.
- CAD/CAM computer-aided design and manufacturing
- HA powder 40% HA powder (Sigma, St Louis, MO, US A) was added to 60% of PCL particles (Solvay, Brussels, Belgium; average Mw ⁇ 40,000, polydispersity index of 1.2), mixed, the above-mentioned femoral head surface without articular cartilage
- PCL particles Solvay, Brussels, Belgium; average Mw ⁇ 40,000, polydispersity index of 1.2
- FDM rapid prototyping-melting deposition
- the pore size inside the bone phase scaffold is (400 ⁇ 20) ⁇ and (200 ⁇ 20) ⁇ in the plane parallel to and perpendicular to the bottom, and the porosity is (54.6 ⁇ 1.2)%.
- the stent is designed with biological and mechanical considerations in mind.
- the biphasic scaffold Before inoculation and in vitro culture, the biphasic scaffold is first soaked in 75% alcohol for 2 hours, then irradiated with ultraviolet light for at least 30 minutes, and precultured for 2-3 days.
- chondrocytes and bone-phase scaffolds were inoculated with chondrocytes and bone marrow-derived stem cells after osteogenic induction.
- P2 chondrocytes were inoculated on PGA/PLA scaffolds at a seeding density of 50 ⁇ 10 6 /ml. The cells are seeded on the scaffold, Incubate for 4-5 hours, then culture the chondrocyte-PGA/PLA scaffold complex for 2-6 weeks (Fig. 4A).
- the culture medium included 10 ng/mL transforming growth factor ⁇ (TGF- ⁇ , InterGen, Burlington, MA), 40 ng/mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL insulin Growth factor 1 (IGF-1; Sigma-Aldrich,
- ITS insulin-transferrin-sodium selenide
- bone marrow stromal stem cells after P2 generation osteogenic induction were inoculated into microtubules inside the PCL/HA scaffold, and the seeding density was 25 ⁇ 10 6 /ml.
- the cells were seeded into tiny tubes inside the scaffold, and after 2-3 hours of incubation, the remaining cells were inoculated again onto the scaffold, incubated again, and 2-3 hours later, the osteogenic induction culture fluid was added for 2-6 weeks (Fig. 4B).
- the bone mass of the section we measured bone volume/total tissue volume (BV/TV) (Fig. 8C), bone surface area/bone volume (BS/BV) (Fig. 8D), total osteoblast surface/total tissue area (Ob. S/T.Ar) (Fig. 8E) and total number of blood vessels/total tissue area (Fig. 8F).
- DAB Diaminobenzidine
- cartilage-like cartilage-like tissue regeneration was observed on the joint surface of the experimental group, and white hard bone-like tissue grew in the tiny tubules of the PCL/HA stent, and the regenerated articular cartilage-like tissue
- the surface is smooth, continuous, non-vascularized, and well integrated with the regenerated bone-like tissue, tightly coupled to each other, and there is no visible crack in the interface between the two.
- the size and shape of the regenerated femoral head are identical to those of the goat's femoral head, and the size is 20x l9x l6mm 3 (long ⁇ ⁇ ⁇ high) (Fig. 4 ⁇ ).
- there was no macroscopic cartilage-like tissue regeneration on the joint surface of the control graft and there was no white hard bone-like tissue in the tiny tubules of the PCL/HA stent, instead of loose connective tissue.
- the tissue-engineered femoral head joint surface was smooth and continuous, and the tissue reddish 0 was positive, suggesting that cartilage tissue occurred in the articular surface of the femoral head in the experimental group (Fig. 5 ⁇ ⁇ 0 0/fast green), and PCL/HA under the cartilage In the tiny tube of the scaffold, the staining of Safranin 0 was negative (Fig. 5B Safranin 0/fast green), indicating that the regenerated cartilage tissue has the same function of secreting aggrecan as normal cartilage tissue.
- the Safranin-positive tissue extended into the Safranin-negative tissue and the two were well integrated (Fig. 5B Safranin 0/fast green).
- Type II collagen is immunolocalized to the extracellular matrix of chondrocytes (Fig. 5A Type II collagen).
- the control group was negative for type II collagen.
- the vast majority of PGA/PLA scaffold materials have been degraded, with only a very small amount of material fibers remaining (shown by the arrows in Figure 5A).
- the control group only a small amount of fibrous connective tissue was grown on the surface of the PCL/HA scaffold material and in the microchannel, and the PGA/PLA scaffold had disappeared.
- the regenerated joint surface cartilage tissue extends to the regenerated subchondral bone portion, and the interface between the two shows no cracks under the naked eye and optical microscopy, and they are well integrated.
- chondrocytes near the subchondral bone tissue showed hypertrophic calcification (Fig. 5B).
- Fig. 6B VO n K OSSa X 10
- the trabecular bone structure is the primary anatomical and functional unit of cancellous bone.
- the regenerated bone tissue has a distinct trabecular structure and a large number of bone cells.
- Columnar osteoblasts are present on the surface of the trabecular bone structure (Fig. 6A Fanhong 0/Quick Green x20).
- Fig. 6A hematoxylin-eosin 40
- Type I collagen, osteopontin, osteocalcin and osteonectin were immunolocalized to the regenerated tissues in the tiny tubes of the PLC/HA scaffold, respectively, which were consistent with the distribution of bone tissue and the von Kossa positive distribution.
- Bone histogenesis and angiogenesis were not significantly different outside, in the middle and inside of the regenerative tissue. New blood vessels are generally close to the area of bone marrow tissue.
- the regenerated cancellous bone has obvious bone marrow-like tissue (Fig. 6A Goldner Tricolor X 40).
- tissue engineering joint biphasic scaffolds based on CAD/CAM technology and normal goat femoral head Fully anastomosed, with internal controlled pore size and porosity; using tissue engineering techniques, composite seed cells form a cell-tissue joint biphasic scaffold construct that regenerates tissue engineered goat femoral heads under the skin of nude mice.
- the regenerated tissue engineering femoral head size and shape match the normal femoral head.
- the regenerated articular cartilage and subchondral bone have the biological and mechanical biomechanical properties of normal tissues, and the interface between the two is well integrated for the future. Joints that perform tissue engineered joint replacement instead of lesions offer a potential for bioreconstruction.
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Abstract
一种组织工程关节双相支架,是由骨相支架和软骨相支架组成,所述得软骨相支架是PGA/PLA支架,所述的骨相支架是PCL/HA支架。一种细胞-组织工程关节双相支架构建物及其制备方法及应用也被提及。组织工程关节双相支架利用CAD/CAM技术制备,可精确控制外部形态和内部结构,使骨相和软骨相匹配,利用组织工程技术复合种子细胞形成细胞-组织工程关节双相支架构建物,在体内再生组织工程关节,为组织工程关节替代人工关节进行生物重建提供可能。
Description
一种组织工程关节双相支架及其制备方法和应用
技术领域
本发明涉及组织工程技术领域, 具体地说, 是一种组织工程关节双相支架及其制备 方法和应用。 这种双相支架是由骨相支架和软骨相支架组成, 分别用于构建骨和关节软 骨并且两者完全匹配。
背景技术
各种关节创伤、 炎症、 肿瘤等疾病影响着世界上数亿人的生活, 并且随着人口的老 龄化以及城市建设和交通的发展, 这一数字还会逐年增加。 上述这些疾病中晚期的治疗 主要是进行人工关节置换手术, 这是一种非生物意义上的治疗。 目前人工关节所使用的 材料主要是金属、 生物陶瓷和超高分子聚乙烯, 在关节使用过程中会不可避免地产生机 械性磨损颗粒, 而这些磨损颗粒可以导致骨溶解, 最终引起人工关节松动导致手术失败; 并且无论怎样提高材料性能和改善加工工艺, 机械性磨损颗粒都是会不可避免产生的, 因此关节松动是迟早的事情。 更糟糕的是, 初次人工关节置换失败后再次翻修的手术失 败率更高。 因此, 有必要进行生物型关节的探索和研究。 虽然克隆技术具有划时代的意 义 ,但是也有其自身不可避免的缺陷, 并且组织或者器官克隆技术远非现在的科技所能 企及, 遥遥无期。 组织工程技术的发展为组织工程关节的再生提供了一种可能, 从理论 上讲在适当的关节支架上接种种子细胞, 辅以细胞因子可以在体内再生组织工程关节, 这是一种真正意义上的生物型关节, 为组织工程关节替代现行人工关节进行生物重建病 损的关节提供了一种可能。
组织工程关节大小和形态应该与正常关节精确匹配, 再生的关节包括软骨和软骨下 骨之间的界面与正常组织结构相似或者一致, 再生组织具有正常组织所具有的生物学以 及机械生物生物力学特性等。但是迄今为止, 组织工程化关节的再生尚没有突破性进展。 发明内容
本发明的目的是针对现有技术中的不足, 提供一种组织工程关节双相支架。
本发明的第二个目的是 , 提供一种组织工程关节双相支架的制备方法。
本发明的第三个目的是 , 提供一种组织工程关节双相支架的应用。
本发明的第四个目的是 , 提供一种细胞 -组织工程关节双相支架构建物。
本发明的第五个目的是 , 提供一种细胞-组织工程关节双相支架构建物的制备方法。 本发明的第六个目的是 , 提供一种细胞-组织工程关节双相支架构建物的应用。 为实现上述目的, 本发明采取的技术方案是: 一种组织工程关节双相支架, 所述的 双相支架是由骨相支架和软骨相支架组成, 所述的骨相支架是聚乳酸 /聚乙醇酸
(PGA/PLA) 支架, 所述的软骨相支架是聚 ε -己内酯 /羟基磷灰石 (PCL/HA)支架。
为实现上述第二个目的, 本发明采取的技术方案是: 所述的组织工程关节双相支架 的制备方法, 其特征在于, 包括以下步骤: 首先激光扫描或者 CT和 MRI扫描, 然后计 算机辅助设计和三维重建关节支架, 并通过计算机辅助制造快速成型技术加工制备骨相 支架, 以及软骨相支架模具, 利用软骨相支架模具加工制备软骨相支架。 骨相支架和软 骨相支架组成组织工程关节双相支架, 其中骨相支架是 PCL/HA支架, 软骨相支架是 PGA/PLA支架。
为实现上述第三个目的, 本发明采取的技术方案是: 所述的组织工程关节双相支架 在制备组织工程关节中的应用。
为实现上述第四个目的,本发明采取的技术方案是: 一种细胞-组织工程关节双相支 架构建物, 所述的细胞 -关节双相支架构建物由成骨部分和成软骨部分组成, 所述的成骨 部分是骨髓基质干细胞 -PCL/HA骨相支架, 所述的成软骨部分是软骨细胞或骨髓基质干 细胞 -PGA/PLA软骨相支架。
所述的成骨部分的制备方法: 采用权利要求 2所述的方法制备 PCL/HA骨相支架, 支架内接种骨髓基质干细胞, 然后进行体外成骨诱导培养 2-6周。
所述的成软骨部分的制备方法: 采用权利要求 2所述的方法制备 PGA/PLA软骨相 支架, 支架内接种软骨细胞或骨髓基质干细胞; 然后体外培养 2-6周。
为实现上述第五个目的,本发明采取的技术方案是:所述的细胞 -组织工程关节双相 支架构建物的制备方法, 其特征在于, 包括以下步骤: (1 ) 采用权利要求 4所述的方法 制备成骨部分, PCL/HA骨相支架内接种骨髓基质干细胞, 然后进行体外成骨诱导培养 2-6周。 (2) 采用权利要求 4所述的方法制备成软骨部分, PGA/PLA软骨相支架内接种 软骨细胞或骨髓基质干细胞; 然后体外培养 2-6周。
为实现上述第六个目的,本发明采取的技术方案是:所述的细胞 -组织工程关节双相 支架构建物在制备组织工程关节中的应用。
本发明优点在于:
本发明组织工程关节双相支架是在基于计算机辅助设计 /计算机辅助制造 (CAD/CAM)技术基础上加工制备, 其外部形态和内部结构都精确可控, 并且骨相和软 骨相支架相匹配, 然后利用组织工程技术, 复合种子细胞形成细胞-组织工程关节双相支 架构建物, 可以在体内再生组织工程关节, 再生的关节软骨和软骨下骨具有正常组织所 具有的生物学及机械生物力学等特性, 并且两者之间界面整合良好, 为将来组织工程关 节替代现行人工关节进行生物重建病损的关节提供了一种可能。
附图说明
图 1 : 组织工程股骨头再生实验流程图
山羊关节软骨细胞的分离、培养、接种到 PGA/PLA支架上和体外培养 (A-B1-C1-D1 ); 骨髓基质干细胞的分离、 培养、 接种到 PCL/HA支架上和体外诱导培养 (A-B2-C2-D2); 上述两种细胞-支架复合物组成股骨头移植物(E);将上述移植物植入裸小鼠背部皮下 10 周 (F )。 山羊股骨头激光扫描, 通过 CAD/CAM 技术, 加工制备 PCL/HA骨相支架 (G-H-I-J);此夕卜, PGA/PLA软骨相支架是通过 CAD/CAM技术制备出的模具压制而成。
图 2: 分 、 B、 C三部分
A部分: PGA/PLA和 PCL/HA的表面形态以及材料的生物相容性
PGA/PLA支架大体观(A1 ); 扫描电镜(χ200), 箭头示 PLA在 PGA纤维上的涂层 及其形成的连接, 这有助于支架形状的维持(A2); 电镜下软骨细胞在支架上的粘附和伸 展以及细胞外基质的形成情况(A3 )。 PCL/HA支架大体观(A4); 扫描电镜(><35 ) (A5 ); 电镜下 (χ500) 骨髓基质干细胞在支架上的粘附和伸展 (A6)。
B部分: 不同含量的 PLA和 HA对 PGA/PLA和 PCL/HA支架特性的影响
随着 PLA含量的增加, PGA/PLA支架的机械生物力学强度在增加, (图 2 Bl), 但 是软骨细胞的接种效率却逐渐下降 (图 2 B2)。
同样地, 随着 HA含量的增加, PCL/HA支架应力应变曲线 (图 2 B3)显示 PCL/HA支 架具有更陡峭的线弹性区域以及压缩力更高的平台区域, 压缩模量 (图 2 B4)和压缩力 (图
2 B5)也相应增高; 吸水率也相应增加 (图 2 B6)。
C部分: PGA/PLA和 PCL/HA支架与正常软骨和松质骨的弹性模量区间范围对比 骨髓间质干细胞 (BMSCs)是一类具有多向分化潜能的成体干细胞,其分化方向与所处 的周围环境包括支架的弹性模量或者硬度有关。 PCL/HA支架弹性模量在正常松质骨弹 性模量区间范围内, 这有助于进一步提高向成骨方向分化能力; PGA/PLA支架的弹性模 量范围在正常关节软骨的弹性模量区间范围内, 这有助于进一步提高向成软骨方向分化 能力。
图 3 : 基于 CAD/CAM技术构建组织特异性双相支架
激光扫描山羊股骨头(A), 扫描数据基于 CAD (B, C)和 CAM技术采用三维打印 技术制备骨相 PCL/HA支架, 其下面包括手术时***骨髓腔固定的柄, 内部有相互连接 的微小管道。骨相支架内部的孔径在平行于和垂直于底部的平面分别是(400±20) μιη和 (200±20) μιη。 (D, E, H)。 PGA/PLA支架则是通过计算机布尔减运算获得软骨相数据, 然后通过上述 CAD/CAM技术三维打印加工制备软骨相支架模具, 通过模具加工制备软
骨相 PGA/PLA支架 (F, 1)。 构建的股骨头双相支架 (G)。
图 4: 细胞 -支架材料复合物光学显微镜下、 肉眼观以及体内植入和标本取材。
关节软骨细胞 -PGA/PLA支架光学显微镜下观, 箭头示细胞接种 2周后在支架上的 粘附和生长情况(A) ; BMSCs-PCL/HA支架光学显微镜下观,箭头示接种 2周后 BMSCs 在 PLC/HA支架的微小管道中的粘附和生长情况(B ); 这两种细胞-支架复合物组合在一 起形成了股骨头移植物 (C); 裸小鼠背部皮下移植物 (箭头示) (D); 10周后在股骨头 移植物的关节表面再生出具有肉眼可见的软骨样组织, 在 PLC/HA支架的微小管道中有 白色质硬骨样组织长入, (E); 股骨头整个关节面部位再生的软骨组织光滑、 连续、 无血 管化并且很好地跟软骨下骨组织整合在一起, 没有肉眼可见的裂隙和分层现象 (F)。
图 5 : 再生的软骨组织以及软骨和软骨下骨界面
软骨 (A): 组织工程化股骨头整个关节表面软骨光滑、 连续, 再生的软骨组织中没 有发现血管长入, 软骨细胞在细胞外基质陷窝中。 组织番红 0呈阳性, 提示实验组中股 骨头关节面有软骨组织发生(图 5A番红 0/快绿), 说明再生的软骨组织具有跟正常软骨 组织同样的分泌聚集蛋白聚糖功能。 II型胶原免疫定位于软骨细胞的细胞外基质 (图 5A II型胶原)。 另外, 绝大部分 PGA/PLA支架材料已经降解, 仅有极少量的材料纤维残留 (箭头示)。
界面 (B ): 再生的股骨头关节表面软骨组织延伸到再生的软骨下骨部分, 两者之间 界面没有裂隙和分层, 它们整合良好。 另外, 在靠近软骨下骨组织的软骨细胞呈肥大钙 化。番红 0阳性组织延伸到番红 0阴性组织中并且两者整合良好(图 5B 番红 0/快绿)。
图 6: 再生的骨组织
常规石蜡包埋切片染色 (A) : 在标准石蜡包埋切片染色处理过程中 PCL/HA支架材 料已经被清除。 再生的骨组织具有明显的骨小梁的结构, 并有大量的骨细胞 (苏木素-伊 红, 番红 0/快绿, 甲苯胺蓝, Goldner三色, >< 10), 骨小梁结构表面有排列生长的柱状 成骨细胞 (图 6Α番红 0/快绿 χ20)。 骨小梁结构是松质骨首要的解剖和功能单位。 极好 的骨组织长入也间接表明 PCL/HA支架材料的降解 (箭头示) (图 6Α番红 0/快绿 χ40)。 另外, 在再生的松质骨组织中有明显的小血管形成 (图 6Α 苏木素 -伊红 χ40)。 再生的 松质骨内具有明显的骨髓组织(图 6Α Goldner三色 χ40)。 可见典型的骨细胞(箭头示) 是一种常见的细胞, 它具有 1-2个核仁(图 6Α甲苯胺蓝 χ40)。 再生的骨组织内有由血管 内皮组成的管腔内的红细胞(箭头示)(图 6Α苏木素 -伊红 χ40)。 高倍光学显微镜下的骨 髓样组织 (图 6Α Goldner三色 X 40)。
聚异丁烯酸甲酯包埋硬组织磨片染色(B ): von Kossa染色显示在 PLC/HA支架相互
连接的三维微小管道中有阳性钙化沉积 (图 6B von Kossax lO); 甲苯胺蓝染色显示有极 好的骨组织生长, 并且具有骨小梁结构, 箭头示 PCL/HA支架材料降解形成的孔隙 (图 6B 甲苯胺蓝 χ 10)。
标尺 x lO: 200 μιη, x20: 100 μιη, x40: 50 μιη
图 7: 再生骨组织部分免疫组织化学成骨的鉴定
在成骨部分示骨桥蛋白阳性。 骨桥蛋白是一种细胞外基质细胞粘附蛋白, 不仅可以 由前成骨细胞, 成骨细胞, 骨细胞生物合成, 而且血管内皮细胞也可以生物合成。 箭头 示再生的血管组织 (Α)。 I型胶原 (Β)、 骨钙蛋白 (C) 和骨连接蛋白 (D ) 也分别阳性 表达, 它们与骨组织的分布和 von Kossa阳性分布区域相一致。 标尺: 200 μιη
图 8: 组织学定量分析
再生的软骨部分: 我们根据国际软骨修复委员会组织学分会的视觉组织学评分标准 进行软骨部分评分 (图 8Α), 测量软骨细胞数 /总的软骨面积 (图 8Β)。 上述定量分析实 验组和正常对照组之间没有显著性差异。 但实验组和对照组存在显著性差异。
再生的骨部分: 我们测量了骨容量 /总的组织容量 (BV/TV) (图 8C)、 骨表面积 /骨 容量(BS/BV) (图 8D)、 总成骨细胞表面 /总组织面积 (Ob.S/T.Ar) (图 8E) 以及总血管 数量 /总组织面积 (图 8F)。 上述所有测量结果在实验组和正常对照组之间没有显著性差 异, 但是在实验组和对照组之间存在显著性差异。
图 9: 机械生物力学测定
再生软骨组织的机械生物力学测定:实验组和对照组在再生软骨组织的压缩模量(图 9A) 和压缩力 (图 9B ) 方面没有显著性差异 (n=10, p>0.05 )。 由于对照组中没有明显 软骨组织再生, 所以实验组和对照组之间存在显著性差异 (n=10, p<0.05 )。
再生骨组织的机械生物力学测定:实验组和正常组在压缩模量(图 9C)和压缩力(图 9D ) 方面没有显著性差异存在 (n=10, p > 0.05) o 但实验组和对照组有显著性差异存在 (n=10, p<0.05)o 实际上, 由于对照组中几乎没有再生的骨组织形成, 所以压缩模量和压 缩力测得的实际上是尚未完全降解的 PCL/HA支架及其内部长入的纤维***的压缩 模量和压缩力, 而不是再生的骨组织的压缩模量和压缩力。
图 10: 对照组组织再生情况
关节双相支架没有软骨和骨组织再生, PCL/HA支架材料表面和微小管道中只有少 量纤维***生长, 上面支架关节面部位的 PGA/PLA支架已经消失, 只有少许纤维结 缔组织生长 (箭头示)。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
本文中所述的 PGA/PLA支架是指 PGA和 PLA复合材料支架, PCL/HA支架是指 PCL和 HA复合材料支架, 也即 "/"是指前后二者为 "和"的关系而不是 "或"的关系。
实施例
1.材料和方法
1.1 总体实验设计
将 20只 8周龄裸小鼠(中国科学院上海实验动物中心)随机分成两组:实验组 (n=10), 对照组 (n=10); 另外把 10个 10月龄的山羊股骨头作为正常对照组 (n=10)。 实验组在裸 小鼠背部皮下植入细胞支架复合物,对照组在裸小鼠背部皮下植入没有复合细胞的支架。 所有动物实验严格遵守上海交通大学医学院关于实验动物使用的管理规定和并征得同 意。 总体实验设计图见图 1。
1.2 材料表征
随着 PLA含量从 0%增加到 30%, 聚羟基乙酸 /聚乳酸(PGA/PLA)支架的压缩模量 逐渐增加,然而软骨细胞接种效率却在逐渐下降。 10%PLA比例的 PGA/PLA软骨相支架 充分平衡了机械力学特性和细胞接种效率。
聚 ε-己内酯 /羟基磷灰石 (PCL/HA)具有良好的生物相容性、生物可降解性、非病原性、 骨诱导活性, 并且 HA也是骨无机物中最重要的组成部分。 PCL/HA支架具有良好的细 胞粘附性。 另外, 随着 HA含量从 10%增加到 40%, PCL/HA支架具有更陡峭的线弹性 区域以及压缩力更高的平台区域, 压缩模量和压缩力也相应增高。 而且随着 HA含量的 增加, 吸水率也相应增加。但 HA含量如果大于 40%,那么快速成型制备支架时 PCL/HA 熔丝就不能喷出, 所以我们选择 40%HA比例的 PCL/HA支架。
1.3 分离和培养软骨细胞以及骨髓基质干细胞
一只 10月龄的山羊作为种子细胞: 软骨和骨髓基质干细胞的来源。 从山羊膝关节股 骨髁间窝处取少许关节软骨组织,切碎, PBS CHyClone, Logan, UT, USA)洗涤,然后 0.1% 胶原酶 (Worthington Inc., Lakewood, NJ, USA)在 37°C摇床 8-12小时,最后 100 μιη滤网过 滤后接种到培养皿, 5% C02, 37°C条件下培养。
同时, 从山羊膝关节胫骨近端髁部抽取骨髓, 分离并接种到培养皿, 细胞接种密度 是 5χ 105单核细胞 /cm2, 5% C02, 37°C条件下培养。 从 P2代开始使用成骨诱导液继续培 养。 其中含有 10mM β-甘油磷酸二钠 (Sigma-Aldrich, St. Louis, MO, USA), Ο.ΙμΜ地塞米 松 (Sigma-Aldrich) and 50μΜ 2-磷酸抗坏血酸 (Gibco)。
1.4 激光扫描
激光扫描仪 (Konica Minolta, Sakai, Osaka, Japan)分别采集有 /无关节软骨的股骨头的 表面形态,分辨率是 8μιη。同样的,我们也可以计算机断层扫描 (CT)或核磁共振成像(MRI) 扫描。
1.5 计算机辅助设计 (CAD)
计算机辅助设计 (CAD) 并三维重建, 骨相支架包括股骨头和一个***髓腔的柄用 来手术***固定在髓腔, 大小是 18x l7x l5mm3 (长 X宽 X高), 与正常山羊股骨头大小和形 态一致。 由于这个尺寸远超过再生组织物质交换的距离 200μιη,所以设计了内部相互连接 的三维微小管道 (直径 200-400μιη;)。
1.6 软骨相 PGA/PLA支架的制备
首先, 计算机辅助设计和制造 (CAD/CAM)技术制备模具。 将上述激光扫描获得的有 /无关节软骨的股骨头表面形态数据, 经布尔减运算获得关节软骨数据, 然后计算机辅助 设计关节软骨支架模具, 全部数据输入到 CAM*** (Spectrum 510, Z Corporation;), 三维 打印制备出树脂模具, 包括内外两部分。 为便于给模具内的材料加压, 外面部分被硅橡 胶所代替。 将 50mg无纺 PGA纤维 (组织工程国家中心, 上海) 置于模具内施压 12小 时制备成关节面外形, 1.0 mm 厚(图 3 F)。 0.3% PLA (Sigma, St. Louis, MO, USA)二甲苯 溶液反复均匀地滴在 PGA支架上, 65°C烘箱干燥, 直到 PLA含量为 10%, 再次用模具 压型, 最后根据模具边缘修剪多余材料。
1.7 骨相 PCL/HA支架的制备
40%HA粉末 (Sigma, St Louis, MO, US A)加入 60%的 PCL 颗粒 (Solvay, Brussels, Belgium; average Mw~40,000, polydispersity index of 1.2), 混匀, 上述无关节软骨的股骨 头表面形态激光扫描数据经计算机辅助设计骨相支架, 数据输入到 CAM***, 120°C加 热熔融, 采用快速成型技术一熔融沉积成型法 (FDM) 制备 (Fortus, Stratasys, Eden Prairie, MN, USA)。 骨相支架内部的孔径在平行于和垂直于底部的平面分别是(400±20) μιη和 (200±20) μιη,孔隙率是 (54.6± 1.2)%。支架的设计充分考虑到了生物学和机械力学因素。
1.8 基于 CAD/CAM技术最终构建的组织工程关节双相支架以及支架的接种前处理 上述 PGA/PLA软骨相支架和 PCL/HA骨相支架组成组织工程关节双相支架, 大小 是 20x 19x 16mm3 (长 χ宽 χ高)(图 3 G)。
在接种和体外培养前, 双相支架先用 75%酒精浸泡 2个小时, 然后紫外线照射至少 30分钟, 预培养 2-3天。
1.9软骨相和骨相支架上分别接种软骨细胞和成骨诱导之后的骨髓基质干细胞 在 PGA/PLA支架上接种 P2代软骨细胞, 接种密度 50x l06 /ml。 细胞接种在支架上,
孵育 4-5小时, 然后软骨细胞 -PGA/PLA支架复合物体外培养 2-6周(图 4A)。 培养液中 包括 10 ng/mL转化生长因子 βΐ (TGF-βΙ, InterGen, Burlington, MA), 40 ng/mL*** (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL*** 1(IGF-1; Sigma-Aldrich,
St. Louis, MO, USA), 1%胰岛素-转铁蛋白 -硒钠 (ITS; Sigma-Aldrich, St. Louis, MO, USA) 等。
同时,在 PCL/HA支架内部的微小管道中接种 P2代成骨诱导之后的骨髓基质干细胞, 接种密度 25x l06 /ml。细胞接种到支架内部的微小管道中, 孵育 2-3小时后将剩余细胞再 次接种到支架上, 再次孵育, 2-3小时后加入成骨诱导培养液体外培养 2-6周 (图 4B)。
1.10 在裸小鼠背部皮下植入细胞支架复合物或单纯支架
常规 1%-5%异氟烷麻醉, 10%聚维酮碘和 70%酒精消毒。 在裸小鼠背部做 20mm长 正中纵行手术切口, 分离皮下组织使之形成一个口袋形腔隙。 细胞-支架移植物分上下两 部分: 上面是软骨细胞 -PGA/PLA支架复合物, 下面是成骨诱导之后的骨髓基质干细胞 -PCL/HA支架复合物, 这两部分组成股骨头移植物 (图 4C)。 单纯无细胞双相支架移植 到裸小鼠背部皮下作为对照组。手术后没有不良反应和并发症。 10周后,麻醉后处死(图 4E)。 标本用 10%中性***固定。
1.11 组织学和组织形态学分析
一部分标本用 0.5M EDTA脱钙后采用标准石蜡包埋组织学切片染色,其余标本直接 聚异丁烯酸甲酯包埋, 30 μιη磨片。 按照随机原则, 前者分别苏木素 -伊红 (HE)染色、 番 红 0-快绿染色、 甲苯胺蓝染色和 Goldner三色染色, 后者分别 von Kossa染色显示钙盐 沉积以及甲苯胺蓝染色。 我们根据国际软骨修复委员会组织学分会的视觉组织学评分标 准进行软骨部分评分, 测量软骨细胞数 /总的软骨面积。 切片的骨部分我们测量骨容量 / 总的组织容量 (BV/TV) (图 8C)、 骨表面积 /骨容量 (BS/BV) (图 8D)、 总成骨细胞表 面 /总组织面积 (Ob.S/T.Ar) (图 8E) 以及总血管数量 /总组织面积 (图 8F)。
1.12 免疫组织化学分析
连续切片经脱蜡、 PBS洗涤后进行抗原修复: 胃蛋白酶 37°C 30分钟, PBS洗涤, 3% ¾02室温下孵育, 然后再次洗涤。 我们分别采用抗 I型胶原单克隆抗体 (1 :200; 5D8, Enzo, Dural NSW, Australia) 和抗 II型胶原单克隆抗体 (1 : 100, ab34712, Abeam, Cambridge, MA, USA) 进行 I型和 II胶原免疫定位; 分别采用骨桥蛋白(1 :200, N/A, Thermo Fisher Scientific, Waltham, Massachusetts, USA)、骨钙蛋白(1 :400, OC4-30, Abeam, Cambridge, MA, USA)和骨连接蛋白(I : 100, ONl-1, Abnova, Taipei City, Taiwan)单克隆抗体进行骨桥蛋白、 骨钙蛋白和骨连接蛋白免疫定位。 分别加入一抗 37°C下孵育 60分钟, PBS洗涤。 然后
室温下用酶共轭的二抗孵育 15分钟, PBS洗涤。 二氨基联苯胺 (DAB)过氧化物酶底物处 理 5-10分钟, 流水洗涤, 苏木素复染 3分钟, 梯度酒精脱水, 二甲苯透明。 阴性对照除 了省略一抗步骤外, 其余过程同上所述。 另外, 正常标本阳性对照。
1.13 再生的软骨和软骨下骨机械生物力学性能测定
我们采用测试*** (Instron, Grove City, PA, USA)进行压缩测试实验。分别在压缩条件 下测试各组软骨和软骨下骨圆柱体 C5.00 [SD 0.10]x5.00[0.20] mm2),应力与应变之比即为 (E)。
1.14 数据分析
采用 SPSS ( 13.0版)进行数据分析, P <0.05 为显著差异。 对于正态分布计数资料, 我们采用一维方差分析和最小显著性差异检验。 对于分布类型未知的资料, 采用非参数 Wilcoxon秩和检验。
2. 结果
2.1 大体观
在裸小鼠背部皮下植入 10周后, 实验组关节表面有肉眼可见的软骨样组织再生, 并 且 PCL/HA支架的微小管道中有白色坚硬的骨样组织长入, 再生的关节软骨样组织表面 光滑、 连续、 无血管化, 并且与再生的骨样组织很好地整合在一起, 彼此紧密结合, 两 者之间的界面没有肉眼可见的裂隙。 再生的股骨头大小和形态与山羊股骨头一致, 大小 是 20x l9x l6mm3 (长 χ宽 χ高) (图 4Ε)。 然而对照组移植物的关节表面没有肉眼可见的 软骨样组织再生, PCL/HA支架的微小管道中也没有白色坚硬的骨样组织长入, 代替的 是疏松***。
2.2 组织工程股骨头关节软骨组织的再生
在组织工程化股骨头关节面部位光滑、 连续, 组织番红 0呈阳性, 提示实验组中股 骨头关节面有软骨组织发生(图 5Α番红 0/快绿),而软骨下面的 PCL/HA支架的微小管 道中番红 0染色呈阴性 (图 5B 番红 0/快绿), 说明再生的软骨组织具有跟正常软骨组 织同样的分泌聚集蛋白聚糖功能。番红 0阳性组织延伸到番红 0阴性组织中并且两者整 合良好(图 5B番红 0/快绿)。 再生的软骨组织中没有发现血管长入, 软骨细胞在细胞外 基质陷窝中。 II型胶原免疫定位于软骨细胞的细胞外基质 (图 5A II型胶原)。 对照组 II 型胶原阴性。 另外, 绝大部分 PGA/PLA支架材料已经降解, 仅有极少量的材料纤维残 留 (图 5A箭头所示)。 在对照组中 PCL/HA支架材料表面和微小管道中只有少量纤维结 缔组织生长, 上面 PGA/PLA支架已经消失。定量分析: 我们根据国际软骨修复委员会组 织学分会的视觉组织学评分标准进行软骨部分评分 (图 8A), 测量总软骨细胞数 /总的软
骨面积 (图 8B)。 上述定量分析实验组和正常对照组之间没有显著性差异。 但实验组和 对照组存在显著性差异。
再生软骨组织的机械生物力学测定:实验组和对照组在再生软骨组织的压缩模量(图 9A) 和压缩力 (图 9B) 方面没有显著性差异 (n=10, p>0.05 )。 由于对照组中没有明显 软骨组织再生, 所以实验组和对照组之间存在显著性差异 (n=10, p<0.05 )。
2.3 再生软骨和软骨下骨之间的界面以及骨再生情况
再生的关节表面软骨组织延伸到再生的软骨下骨部分, 两者之间界面肉眼和光学显 微镜下没有看到裂隙, 它们整合良好。 另外, 在靠近软骨下骨组织的软骨细胞呈肥大钙 化 (图 5B)。
2.4 组织工程化股骨头松质骨再生和血管形成
组织学切片染色显示在 PLC/HA支架的微小管道中有明显骨组织长入, 支架材料只 有极少的降解, 绝大部分尚未完全降解, Von Kossa染色显示在 PLC/HA支架相互连接的 三维微小管道中有阳性钙化沉积 (图 6B VOn KOSSaX 10)。 骨小梁结构是松质骨首要的解 剖和功能单位。 再生的骨组织具有明显的骨小梁的结构, 并有大量的骨细胞, 骨小梁结 构表面有柱状的成骨细胞 (图 6A番红 0/快绿 x20)。 另外, 在再生的松质骨组织中有明 显的小血管形成(图 6A苏木素 -伊红 χ40)。 I型胶原、骨桥蛋白、骨钙蛋白和骨连接蛋白 分别免疫定位于 PLC/HA支架的微小管道中再生的组织, 它们与骨组织的分布和 von Kossa阳性分布区域相一致。骨组织发生和血管形成在再生组织的外部、中间和内部没有 明显差异。 新生血管一般靠近骨髓组织的区域。 再生的松质骨内具有明显的骨髓样组织 (图 6A Goldner三色 X 40)。在对照组中没有新生骨组织形成, 只发现 PLC/HA支架材 料的降解和纤维***生长 (图 10)。 定量分析: 我们测量了骨容量 /总的组织容量 (BV/TV) (图 8C)、 骨表面积 /骨容量 (BS/BV) (图 8D)、 总成骨细胞表面 /总组织面积 (Ob.S/T.Ar) (图 8E) 以及总血管数量 /总组织面积 (图 8F), 上述所有测量结果在实验 组和正常对照组之间没有显著性差异, 但是在实验组和对照组之间存在显著性差异。
再生骨组织的机械生物力学测定:实验组和正常组在压缩模量(图 9C)和压缩力(图 9D ) 方面没有显著性差异存在 (n=10, p>0.05) o 但实验组和对照组有显著性差异存在 (n=10, p<0.05)o 实际上, 由于对照组中几乎没有再生的骨组织形成, 所以压缩模量和压 缩力测得的实际上是尚未完全降解的 PCL/HA支架和其中长入的纤维***的压缩模 量和压缩力, 而不是再生的骨组织的压缩模量和压缩力。
3. 结论
基于 CAD/CAM技术构建的组织工程关节双相支架大小和形态与正常山羊股骨头完
全吻合, 并且具有内部可控的孔径和孔隙率; 利用组织工程技术, 复合种子细胞形成细 胞 -组织工程关节双相支架构建物, 在裸小鼠背部皮下可以再生出组织工程山羊股骨头。 再生的组织工程股骨头大小和形态与正常股骨头相匹配, 再生的关节软骨和软骨下骨具 有正常组织所具有的生物学及机械生物力学等特性, 并且两者之间界面整合良好, 为将 来进行组织工程关节置换替代病损的关节提供了一种生物重建的可能。
以上所述仅是本发明的优选实施方式, 应当指出, 对于本技术领域的普通技术人员, 在不脱离本发明方法的前提下, 还可以做出若干改进和补充, 这些改进和补充也应视为 本发明的保护范围。
Claims
1.一种组织工程关节双相支架,其特征在于,所述的双相支架是由骨相支架和软骨相 支架两部分组成, 所述的骨相支架为 PCL/HA支架, 软骨相支架为 PGA/PLA支架。
2.根据权利要求 1所述的组织工程关节双相支架的制备方法,其特征在于,包括以下 步骤: 首先激光扫描或者 CT和 MRI扫描; 然后计算机辅助设计和三维重建组织工程关 节支架; 最后通过计算机辅助制造快速成型技术加工制备骨相支架, 以及软骨相支架模 具; 利用软骨相支架模具加工制备软骨相支架; 骨相支架和软骨相支架组成组织工程关 节双相支架, 其中骨相支架是 PCL/HA支架, 软骨相支架是 PGA/PLA支架。
3.根据权利要求 1所述的组织工程关节双相支架在制备组织工程关节中的应用。
4.一种细胞 -组织工程关节双相支架构建物, 其特征在于, 所述的细胞-关节双相支架 构建物由成骨部分和成软骨部分组成, 所述的成骨部分是骨髓基质干细胞 -PCL/HA骨相 支架, 所述的成软骨部分是软骨细胞或骨髓基质干细胞 -PGA/PLA软骨相支架。
5.根据权利要求 4所述的细胞 -组织工程关节双相支架构建物, 其特征在于, 所述的 成骨部分的制备方法: 采用权利要求 2所述的方法制备 PCL/HA骨相支架, 支架内接种 骨髓基质干细胞, 然后体外成骨诱导培养 2-6周。
6.根据权利要求 4所述的细胞 -组织工程关节双相支架构建物, 其特征在于, 所述的 成软骨部分的制备方法: 采用权利要求 2所述的方法制备 PGA/PLA软骨相支架,支架内 接种软骨细胞或骨髓基质干细胞; 然后体外培养 2-6周。
7.根据权利要求 4所述的细胞-组织工程关节双相支架构建物的制备方法, 其特征在 于, 包括以下步骤: (1 )采用权利要求 4所述的方法制备成软骨部分, PGA/PLA软骨相 支架上接种软骨细胞或骨髓基质干细胞; 然后体外培养 2-6周; (2) 采用权利要求 4所 述的方法制备成骨部分, PCL/HA骨相支架上接种骨髓基质干细胞, 然后进行体外成骨 诱导培养 2-6周。
8.根据权利要求 4-6任一所述的细胞 -组织工程关节双相支架构建物在制备组织工程 关节中的应用。
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