TW201136622A - Composite material used for repairing cartilage tissue and preparing method thereof - Google Patents

Abstract

This invention relates to a composite material used for repairing cartilage tissue and preparing method thereof, the composite material comprising: a composition wrapped with cells, containing human Placenta-derived mesenchymal cells and an alginate, the cell-wrapped composition utilizing a transforming growth factor-<beta>, (TGF-<beta>3), to induce the human Placenta-derived mesenchymal cells differentiating into cartilage cells. Furthermore, the cell-wrapped composition can further contain a PGD-containing peptide like CBD-RGD, and biomedical porcelain particles like nano-calcium deficient hydroxyapatite (nCDHA). The TGF-<beta>3 can be contained in the cell-wrapped composition or absorbed in the nCDHA. The composite material can further comprise a PLGA precision scaffold. Utilizing the cell-wrapped composition or combining the cell-wrapped composition with the PLGA precision scaffold, the human Placenta-derived mesenchymal cells can be induced by TGF-<beta>3 to disintegrate into cartilage cells and secret a great amount of extracellular matrix like Glycosaminoglycans (GAG) and Collagen Type II, so as to achieve the objective of repairing cartilage tissue.

Description

201136622 VI. Description of the Invention: [Technical Field] The present invention relates to a biomedical material for cartilage tissue engineering&apos;, particularly to a composite material for repairing cartilage tissue. [Previous technique] Cartilage is a non-vascular, nerve-free connective tissue, mainly composed of extracellular matrix.

(extracellular matric) and a small amount of chondrocytes, which consist of 8〇% collagen (c〇llagen) and 20% protein proteoglycans, in which collagen Type II collagen is predominant. When cartilage tissue is defective due to disease or other causes, its ability to repair itself is limited, so it can be manufactured by cartilage tissue engineering, using chondrocytes in combination with synthetic substrates or natural substrates. The active and functional tissue replaces the diseased tissue in the human body and fundamentally solves the dysfunction caused by cartilage tissue defects. The three elements of cartilage tissue engineering are cells, scaff〇ld, and message factors. Among them, the stent can be regarded as an artificial extracellular matrix, providing a stereoscopic space and sufficient mechanical strength to maintain the space required for the attachment of chondrocytes, allowing the chondrocytes to function normally in the stent and regulating the behavior, behavior and response of the filaments. , in the appropriate day to give the correct signal factors for chondrocytes such as growth factors, paste or differentiation factor chemical factor ship, can promote the activation of chondrocytes or the re-start of the job, and the purpose of the repair . The cell source mainly consists of the same kind of autologous (aibgeneie_group, (Xen〇geneiC)' clinically the most widely used is the extraction of cartilage cells from the normal cartilage block of the patient's. It has the advantages of no immune rejection and disease transmission. However, during the process of cultivating, the money has changed, so that the soft and fine technology is differentiated (dediff_ method holds the score (4) type II collagen, causing the problem of chondrocytes to grow; in addition: there are only hundreds of thousands of autologous chondrocytes, in vitro Cultivate Chichi $2 and the area of autologous chondrocyte transplantation is limited. Fan Fanda is also insufficient, and the source of cartilage tissue can be divided into synthetic materials and natural materials. Among them, the synthetic materials have good mechanical properties and can regulate S. (4), but their cell miscellaneous is worse than natural (four); Tianlu material has excellent cytogenetic f and special biological activity can help chondrocyte growth and secretion matrix. However, it has disadvantages such as excessive degradation and poor mechanical strength. In addition, the conventional stent preparation method has an uncoordinated manufacturing process, no killing, use of a hok forming agent, The shortcomings of the shape limitation, greatly limit the applicability of the stent to the cartilage tissue engineering, and affect the effectiveness of the tissue repair. In order to achieve the best secret effect, the cartilage, the mechanical properties, the biological safety and the phase need to be considered. Factors such as tolerance and message factor, and each factor needs to be compatible with each other. Therefore, Luyou can develop biomedical materials that combine appropriate cells, scaffolds and message factors, which will contribute to the clinical repair of cartilage tissue. SUMMARY OF THE INVENTION In order to solve the problems and disadvantages caused by traditional cartilage tissue engineering, the object of the present invention is to provide a composite material for repairing cartilage tissue, comprising: a cell-coated composition comprising a human placental mesenchymal stem cell (human Placenta-derived mesenchymal cells) and a brown alginate. Another object of the present invention is to provide a method for preparing a composite material for repairing cartilage tissue. The method comprises the steps of: mixing a human placental mesenchymal stem cell ( Human Placenta_derived mesenchymal cells) with a alginate to form a cell-coated component In the composite material and the preparation method thereof, the human placental mesenchymal stem cells can be induced to differentiate into a sacral cell by a transforming growth factor, wherein the transforming growth factor-β is a transforming growth factor•β3′ The transforming growth factor-β may be contained in a composition coated with cells. The composite material further comprises a polylactic acid-polyglycolic acid (PLGA) precision scaffold. In the preparation method of the composite material, The method further comprises combining the cell-coated composition with a polylactic acid-polyglycolic acid (PLGA) precision scaffold. IS] 4 201136622 Further, the cell-coated composition may further comprise a RGD-containing peptide and a biomedical ceramic particle having an RGD (Arginine-Glycine-Aspartate) attachment sequence. The peptide having the RGD attachment sequence may be a cellulose binding functional region of the amino acid sequence-RGD attachment sequence (CBD-RGD); the biomedical ceramic particle may be a nano-nano-hydrogen structure. Nano-calcium deficient hydroxyapatite), which is a biocompatibility ceramic in bioceramics, and the transforming growth factor_β system can be adsorbed to biomedical ceramic particles. In the composite material for repairing cartilage tissue and the preparation method thereof, the concentration of each component of the cell φ outer matrix secretion and the repair effect is respectively: the concentration of CBD-RGD is 10 mg/g, and the human placenta is The concentration of leaf stem cells is 丨M〇6cells/scaff〇w, the concentration of chloroform-filled chloroapatite is 50-1000 ppm, the concentration of alginate is 1.2%, and the concentration of transforming growth factor is 0.01 μ§ /μ1. In addition, the PLGA precision scaffold is prepared by liquid-frozen deposition manufacturing (LFDM), and the PLGA precision scaffold has a nozzle aperture of 0.2 mm, and the interfiber spacing (intervalbetween adjacent fibers) is 0.7 mm. On the other hand, when the composite material for repairing cartilage tissue of the present invention induces differentiation, the transforming growth factor 屮3 (concentration is about 10 ns/m丨) can be further added to the chondrogenic induction medium. In order to promote differentiation of human placental mesenchymal stem cells into chondrocytes, and differentiation induced by 21 days can produce about 6pg/scaff〇|d type II collagen and about 80 pg/scaffo丨d glucosamine polymerized sugar, thereby The secretion of extracellular matrix achieves the purpose of chondrocyte regeneration and repair of damaged cartilage tissue. The present invention will be described in conjunction with the embodiments of the present invention, and the present invention is not intended to limit the scope of the present invention, and those skilled in the art, without departing from the spirit and scope of the present invention. In the meantime, the scope of protection of the present invention is defined by the scope of the appended claims. [Embodiment] The composite material for repairing cartilage tissue of the embodiment of the present invention is a cell-coated group 201136622, and can further be combined with a PLGA precision scaffold, and then using transforming growth factor _p (tmnsf〇rming gmwthfaCt) 〇rp, TGF-p), for example, TGF-h induces chondrogenesis and differentiation. Wherein, the cell-coated composition comprises stem cells and alginate, and further comprises a RGD-containing peptide, such as CBD-RGD, and transforming growth factor-β. It can be adsorbed to biomedical ceramic particles such as nano-calcium deficient hydroxyapatite (hereinafter referred to as nCDHA) or contained in the cell-coated composition. The composite material for repairing cartilage tissue according to the embodiment of the present invention confirms the induction of chondrogenesis by analyzing the amount of chondrocyte production and the extracellular matrix produced by chondrocytes: proteoglycan and type 2 collagen. Chondrogenesis) and the effect of differentiation. Among them, glucosamine-polymerized sugar (GAG) is the main component in protein polybilirubin. Therefore, the analysis of protein poly-audio is based on the detection of glucosamine-polymerized sugar (GAG). The analytical method includes Al-Blue staining. (Aician Blue stain), Hematoxylin-eosin (H&amp;E) and glucosamine polymeric sugar analysis; type II collagen expression was analyzed by immunofluorescence staining with type II collagen And enzyme linked immunosorbent assay (ELISA) analysis. In the analysis of the amount of chondrocyte production, the induced differentiation of chondrocytes is dried by freeze-drying method, and then 1.5 ml of the decomposition liquid is added (the decomposition liquid contains 55 mM of trisodium citrate dehydrate, Showa). 150 mM sodium chloride, # Sigma and 5 mM cysteine acid [CySteine HC1, Sigma] and 5 mM ethylenediaminetetraacetic acid [Na^DTA, Sigma], used Before taking 1 mg of papaya enzyme [papain, Sigma] dissolved in 21 ml of the decomposition solution), placed at 6 〇. (: The reaction was carried out for 24 hours. The cell liquid was adjusted to 6 concentration gradients as a standard by decomposing liquid; the sample of chondrocyte liquid of 〇·5 ml was taken out and the standard product was added to the fluorescent dye of 5〇11. ^1^33258 dye [8丨§1^] was added to Tris-HCl containing 10 福 [Riedel-de Hagn®, Germany], 1 mM disodium edetate [NafDTA, Sigma], 0.1 In mM sodium carbonate buffer solution, adjust ρΗ=7·4, the final concentration of the dye is 0.1 gg/ml), mix well and let stand for 1 hour in the dark. Determine with a fluorescence photometer (F2500, Hitachi) Absorption value (Em: 458nm, Ex: 365 nm) 'The calibration curve is prepared from the standard, and the number of chondrocytes is quantified by this calibration line. 201136622 In the case of tissue section preparation and staining, the induced cells are 10%. After fixing for 48 hours, the embedding cassette was placed in an embedding cassette for gradient alcohol dehydration, dimethyl substitution and paraffin infiltration, and then paraffin-embedded to obtain sample wax. Using a slide slicer, 5 #111 thick sections were cut with a tissue microtome, dewaxed with xylene, and then gradient The fine solution is hydrated, and the test piece can be subjected to Alcian Biue stain and hematoxylin-eosin staining (out 11^0\&gt;^11-605丨11,11&amp;£). The dyeing principle of the indigo blue dyeing method is based on Alcian blue, which is a staining agent for dyeing glucanamine-polymerized sugar (glyC〇lsamin〇glycan, GAG) into blue, and the glucosamine-polymerized sugar (GAG) It is the main component in proteoglycans. It is dyed by first dissolving 1 gram of sigma in 3% acetic acid aqueous solution and adjusting the pH to Μ. The induced cell tissue sections are immersed in Alcian. Shoot for 3G minutes, then rinse with running water for 2 minutes, then put it in Q1% nuclear solid red (Qing 1 from coffee) dye for 5 minutes, rinse with running water for 1 minute, and then put it in 8〇%9〇% The reaction was carried out for 3 minutes with 100% alcohol solution, and the dehydration step was carried out. Finally, it was placed in a solvent of xylene, sealed with a water-insoluble sealing gel, and the staining result was observed by an upright optical microscope. Dyeing is a commonly used staining method for histopathology. Sumu Zi (the wrist of the wrist can dye the nucleus into a blue color) For example, the cytoplasm can be stained pink to observe the tissue structure and cell shape. The induced cell tissue sections are immersed in xylene solvent for 5 minutes, the excess sarcophagus is dissolved, and the hydration is tilted. In 100%, 90%, 8G% and 70% #罐 solution, each soak for 30 seconds, washed with running water for 1 minute, remove excess water, placed in a solution of Merck (Germany) for 2 minutes, for the nucleus Dyeing, and then clearing by running water for a minute, placed in Yihong (Sigma, Canada) solution for 1 minute, cytoplasmic staining, sequentially placed at 7〇%, 80. /. 90. /. 95% with 腑. The alcohol solution was quenched for a minute, and the dehydration step was finally placed in a xylene-soluble needle, and the film was sealed with a water-insoluble sealing gel, that is, the dyeing was completed and the staining result was observed with an upright optical microscope. The method of glucosamine polymerization analysis comprises the step of adding cold cells to the cells, adding 5 ml of the decomposing solution as described above, and reacting at 6 Torr for 24 hours. Prepare the standard of ch〇ndr〇tin 6-sulfate C sodium salt (Sigma) with the decomposition solution as the solvent; take the sample and standard of α5 mi, add 5 mi DMMB (1,9-Dimethyl-methylene blue) dye ,well mixed. The absorption value was measured at a wavelength of 525 nm using a visible light/ultraviolet light 201136622 spectrometer (U200, Hitachi, Japan), and the calibration curve was drawn by the standard result, and the GAG content in the sample was quantified by this calibration line. Immunofluorescence staining analysis of type II collagen utilizes immunochemical staining reagents to bind type 2 collagen with immune antibodies and to detect type II collagen in the extracellular matrix of cartilage by the presence of green fluorescence. The performance of the protein. This method consisted of placing the induced cell tissue sections in a 70 C oven for 15 minutes to adhere the tissue sections to the slides. Soak the tissue section in the solvent of diphenylbenzene for 5 minutes, dissolve the excess paraffin, and then carry out the hydration step. The test pieces are sequentially placed in 100%, 95%, 90%, 80%, 70% alcohol solution, respectively. After soaking for 5 minutes, after washing with phosphate-buffered saline (PBS), the test piece was immersed in 3% H202 for 15 minutes, washed with PBS for 5 minutes; 2% BSA reaction was dripped on the test piece. After 30 minutes, after washing with PBS; drip the type II collagen on the delta plate (M〇use anti human type II collagen

Monoclonal antibody, Neomarkers, Fremont, CA, USA, the first-order anti-system is diluted with PBS at a ratio of 1:100) for 1.5 to 2 hours, washed with pbs; the second antibody of type II collagen is dripped on the test piece. (fluoresceinisothiocyanate (FITC)-conjugated anti-mouse antibody, Gt x Ms IgG Fluor, Temecula, CA, the secondary anti-system is diluted 1:500 in PBS) for 1 hour, washed with PBS; The sealing gel is sealed, that is, the dyeing is completed. The staining results are observed by an upright optical microscope, wherein the positive staining is a green fluorescent reaction, which represents the secretion synthesis of the second type collagen. The content of the second type collagen was analyzed by an ELISA assay kit (ASB-5000-EX, RheumeraTM, Japan), and the experimental procedure was carried out in accordance with the set of tissue manipulation manuals. The above analysis results are expressed as mean ± standard deviation, and t-test (t_test) is used for statistical data analysis'. When the p-value is less than 〇.〇5, there is a statistically significant difference. Through the analysis, the results show that the composite material for repairing cartilage tissue of the embodiment of the present invention, after 21 days of optimal induction time, the number of chondrocytes grown, glucosamine polymerization gall (GAG) and type II collagen The secretion amount of the extracellular matrix is higher than that of the experimental group including only the human placental mesenchymal stem cells and the PL GA precision scaffold and the human placental mesenchymal stem cells, the alginate and the PLGA precision scaffold; and the tissue section staining, the present invention EXAMPLES Characterization of mature cartilage of composite materials used to repair cartilage tissue: 陷acunae is also more pronounced and completes 201136622. In addition, the glucosamine polymerization (gag) of human placental mesenchymal stem cells in the composite material for repairing cartilage tissue of the embodiment of the present invention is significantly more than the human bone marrow mesenchymal stem cells, and thus the embodiment of the present invention is used for repairing cartilage. The composite material of the tissue can be applied to bone tissue engineering to effectively achieve the purpose of cartilage tissue repair. Example 1 Preparation of a cell-coated composition First, a stem cell having a cell density of lyio6 cells/ml was uniformly mixed with an appropriate amount of CBD-RGD. On the other hand, an appropriate amount of nCDHA was added at a concentration of 〇.〇1 pg/ The mixture of mi and tetragen was uniformly mixed. The mixture was placed in a 37 ° C, 5% CO 2 incubator for 1 hour to allow nCDHA to adsorb Lu TGF-β3. The two mixtures were mixed and then added to the alginate solution. A cell-coated composition comprising stem cells, a CBD-RGD protein 'nCDHA adsorbed with TGF-p3, and alginate was obtained. The preparation and analysis of each component in the cell-coated composition are as follows: (1) Stem cell culture, surface protein analysis and induced chondrogenic differentiation analysis The cell-coated composition of the embodiment of the present invention is Human bone marrow mesenchymal stem cells (hBMSCs) or human placenta-derived mesenchymal cells (hPDMCs) are used as cell sources. A. Stem cell culture The placenta of the healthy human mother after delivery in 38-40 weeks of pregnancy is cut into small pieces and washed with PBS to 0.25 ° /. The trypsin enzyme was digested at 37 ° C for 10 minutes and the primary cells of human placental mesenchymal stem cells were isolated. On the other hand, the bone marrow tissue is obtained from the intestinal bone of a 45-year-old patient, and after appropriate treatment, the primary cells of the human mesenchymal stem cells are obtained. The human primary placental mesenchymal stem cells and the primary cells of human bone marrow mesenchymal stem cells were separately cultured in a 75 T-flask (Falcon®, BD Biosciences 'USA) culture dish, which was placed with 880 ml/L DMEM low sugar medium. (Dulbecco's Modified eagle's Medium, low glucose, Gibco), 100 ml/L Fetal bovine serum (FBS, HyClone), 10 ml/L Penicillin-streptomysin (Biological Industries) and 10 The concentration of ml/L is 200 mM L-Glutamine (Tedia). When the primary cells were cultured to a cell density of 8 minutes, they were cultured in 0.25% trypsin/EDTA (Gibco/BRL 'Grand Island, 201136622 NY, USA) and placed in a 37 ° C, 5% C02 cell incubator. Subsequent, cells with a substitution number of 5 to 12 passages were subjected to subsequent experiments. In addition, in order to facilitate the differentiation of stem cells from different individuals used in subsequent experiments, the human bone marrow mesenchymal stem cell line from one patient was given the number of C1008. The number of HSC-1 was given to the human bone marrow mesenchymal stem cell line from another patient. At the same time, the human placental mesenchymal stem cell line from one patient was given the number C081127, and the human placental mesenchymal stem cell line from another patient was given the number C080520. B. Surface protein analysis of stem cells and induction of chondrogenic differentiation analysis In the present invention, the surface marker characteristics of the stem cells were analyzed by flow cytometry. First, human placental mesenchymal stem cells (No. C081127) and human bone marrow mesenchymal stem cells (No. C1008) were removed from the culture plate, and the cell concentration was 5 X 1〇5 cells/mi, and then washed 3 times with pbs. The cell mass was collected by centrifugation; after adding 1 μM of PBS back to the cell mass, 8 μM of fluorescent antibody was added to the cell solution: 匸〇34, CD44, CD45, CD90, CD105, CD106, CD166-C〇 njUgated fluoresceinisothiocyanate FITC, CD29, CD31 and CD73-conjUgated phycoerythrin 'protected from light for 30 minutes, then washed 3 times with PBS, removed unreacted antibody, centrifuged to collect cell mass, and finally added 5 PBS... Block, the above steps were kept at 4 °C. Next, using flow cytometry (FACScan, BD, USA) analysis, cells containing only fluorescent cells were used as background, and antibody-containing cells were used as a control group to determine the cell surface protein expression cluster of differentiation. Marker, CD marker ), the results are shown in the first figure. 0 The results of the cell surface protein expression differentiation group show that human placental mesenchymal stem cells (C081127) and human bone marrow mesenchymal stem cells (cl〇〇8) CD29, CD44, CD73, 3 showed a positive reaction with CD105 (as shown in the first figure), while CDY, cD34, CD45, and CD166 of these stem cells were negative (results not shown), and the results showed that human placental mesenchymal stem cells and humans Bone marrow mesenchymal stem cells can express the same expression of the differentiation group marker (CDmarker). C. Stem cell-induced chondrogenic differentiation production assay To confirm that the stem cells prepared in the examples of the present invention have cartilage differentiation ability, the stem cell culture is carried out by using a chondrium-inducing medium containing 201136622 TGF-β3 growth factor. Extracellular matrix staining was performed 3 weeks after differentiation culture. In the examples of the present invention, the ability of stem cells to differentiate into chondrocytes was induced by using a transwell and the aforementioned staining analysis. Place 2 μμμ of human placental mesenchymal stem cells (C081127) containing about 4 ^ &lt; 1〇5 cells in a 24-well (24_weU, pore size 〇4 _) transfer plate (transwell) made of a polyester material. In the 'transweu' (transweu) at 37. Incubate for 4 hours in a sputum incubator to accumulate cells to form cell clumps, and add excess (that is, the amount of film to be added beyond the bottom of the transfer plate) to induce cartilage differentiation to produce cartilage differentiation medium containing DMEM culture medium (Dulbetxo's Modified Eagle's Medium, Gibco), TGF-p3 (CytoLab/PeproTech, Rehovot, Israel) at a concentration of 1 ng/ml, corticosteroid (Dexamethasone, Sigma) at a concentration of 10 ng/ml, concentration of 50 pg/ml L-Proline, L-Proline, L-Ascorbate-2-phosphate (Sigma), L-Proline (L-Proline, 40. pg/ml)

Sigma) and Insulin-Transferrin-Selenium premix (100X, Sigma) at a concentration of l〇μΐ/mi, cultured for 21 days, and cultured every three days during culture. The solution in which 2TGF_p3 (1〇ng/m丨) is added to the medium has the purpose of maintaining osmotic pressure and achieving uniform differentiation. Next, the cells induced by the culture for 21 days were subjected to Alcian Blue stain and immunofluorescence staining analysis of the second type collagen, respectively, and the results were as shown in the second A and B images and the third A. As shown in the three-B diagram. In the early stage of chondrocyte differentiation, mesenchymal cells aggregate to form a cell cluster containing a small amount of extracellular matrix, which is called biastema; closely arranged germplasm cells begin to secrete extracellular cells. The matrix, the collagen fibers in the extracellular matrix are surrounded by an basophilic substance. The cells in this period are called chondroblasts; the chondrocytes secrete the extracellular matrix to coat themselves, and finally a large number of extracellular matrices. The cell regions are separated to form a space called a lacunae. Please also refer to the second A and B diagrams, which are respectively used in the composite material for repairing cartilage tissue, and the human placental mesenchymal stem cells are differentiated by the cartilage differentiation agent containing transforming growth factor (TGF_p3). A graph of the results of the analysis of the indigo blue staining of chondrocytes at different magnifications. As can be seen from the figure, there are many filamentous fibers, accumulation of glucosamine polymeric sugar (GAG) 201136622, cell (four) episodes, and craters (kcuna) formed by the cell's secretory extracellular group f. Human placental mesenchymal stem cells have the ability to differentiate cartilage. Please also refer to the third continuation of the third level, which is the combination of the human placental mesenchymal stem cells and the cartilage differentiation agent containing transforming growth factor (TGF_p3) into the cartilage. A graph showing the results of immunofluorescence staining analysis of different second-phase proteins of the cells. A green fluorescence distribution can be observed in the figure, and the distribution range is not uniform. The fluorescence intensity near the surface of the sphere is stronger than that in the center, and the expression of the second type collagen can be observed, showing the human placenta mesenchymal Stem cells have the ability to differentiate cartilage. (2) RGD (Arginine-Glydne-ASpartate) attached sequence peptide (RGD-containing peptide) The RGD (Arginine-Glycine-Aspartate) attached sequence (RGD-containing peptide) promotes cell attachment With growth. In the examples of the present invention, the fibrin-binding functional region-RGD attachment sequence (CBD-RGD) protein was tested. The RGD (Arginme-Glycine-Aspartate, RGD) attachment sequence is the smallest functional unit that promotes cell attachment in fibronectin, which promotes cell attachment and maintenance of cell-cleavage-cellulose binding functional regions (cellul 〇se binding domain, CBD) is a sequence fragment of 36 amino acids in the N-terminus of a cellulolytic enzyme (CeliH), a cellulose hydrolase of the fungus (Tricoderma konigii), and has cellulose Affinity, the cellulose-binding functional region (CBD) of the embodiment of the present invention is further stepped into the fifth amino acid acid code of the gene by the polymerization enzyme chain reaction dyme plus reaction, PCR). In order to increase the affinity of the cellulose-binding functional region (CBD) for cellulose. The RGD attachment sequence is then combined with an amino acid-modified cellulose binding functional region (CBD) to form a bifunctional CBD-RGD protein that promotes cell adsorption onto the cellulose, and the protein has SEQ ID NO :l shows the amino acid sequence with a molecular weight of 25 kDa. (3) Bioceramics, hydroxyapatite, calcium-deficient hydroxyapatite (CDHA), nano-calcium hydroxyapatite (nCDHA), etc. Ceramic granules, which contain elements such as calcium (Ca) and phosphorus (P) that can be converted and converted by the body's normal metabolism, or contain hydroxyl groups (-〇) that can bond with human tissue. H) and the like, which have good biocompatibility and properties such as degradation in vivo, and can be used to adsorb proteins. In the examples of the present invention, the CBD-RGD protein was adsorbed by nano-calcium-deficient calcium-depleted ash (nCDHA). nCDHA is a biochemical biochemical (bi〇ceraniics) material with a chemical structure of Ca^PC^KHPC^OHh-, 〇&amp; $ morph length 20-80 nm, diameter 5_1 Corenm core-shell structure, nCDHA has a ratio of calcium to phosphorus (Ca/P) of 1.5, which is the same as the mineral composition and structure of bone tissue, and has good biocompatibility; and the specific surface area of nCDHA Large, can be attached to cells. In addition, nCDHA has the property of adsorbing and slowly releasing proteins such as growth factors, protein drugs and the like and can achieve a specific therapeutic effect by a growth factor or a drug releasing φ mechanism, and in the embodiment of the present invention, nCDHA is adsorbed with TGF- The p3 growth factor, when the nCDHA is combined with the stem cells coated with brown algae, can release TGF-β3 through diffusion and directly induce differentiation into stem cells. Further, in the examples of the present invention, the concentration of nCDHA which can effectively adsorb protein is 5 〇 1 〇〇〇 ppm, preferably 5 〇〇 ppm. (4) TGF-p transforming growth factor stem cells need to add growth factors related to cartilage development to induce cell differentiation toward cartilage. The purpose is to simulate the microenvironment of chondrocyte development and growth in vitro. Differentiation effect. TGF side such as TGF-β], TGF-β2 and TGF-β3 have two-way regulation of chondrocyte differentiation and function 'promoting DNA synthesis and extracellular matrix synthesis of undifferentiated or early differentiated chondrocytes' can also inhibit The role of interleukins, slowing down the catabolism of the cartilage matrix, increased the expression of matrix inhibitors (tissue inhibits 〇f metaU〇piOteinases, Ding), and inhibited the performance of matrix gold layer proteases to improve repair capacity. In the examples of the present invention, the tgf-shabu concentration which is tested at TGF% and which can effectively promote chondrocyte differentiation is 0_1 to 10 ng/ml, preferably i〇ng/mi. (5) Alginate Alginate is a normal chondrocyte body that provides a low-cytotoxic coating effect to maintain the stereotactic configuration, activity and functionality of the cells. In the embodiment of the present invention, the stem cells are coated with brown gum to maintain the globular stereoscopic morphology of the cells, and then differentiate, and the growth factor can be coated to promote the growth of the chondrocytes and maintain the phenotype of the chondrocytes. 13 TS] 201136622 Take 0.8775 grams of sodium sulphate (Sigma) and 1.2 grams of sodium alginate (Hanawa, Japan 'molecular weight 110,000, M / G = l / 2), dissolved in 1 〇二次mi in the second water 'stirred to completely dissolve; sterilized by high temperature and high pressure method, the 丨. 2% alginate solution was cooled and stored at 4 ° C for use. Example 2 Effect of different concentrations of CBD-RGD protein in a cell-coated composition on the induction of chondrogenesis To find an appropriate concentration of CBD-RGD protein added, the present invention analyzes a composition coated with cells. Effects of different concentrations of CBD-RGD protein on stem cell-induced chondrogenesis. Please refer to Table 1 for the human bone marrow mesenchymal stem cells (C1008) and human placental mesenchymal stem cells (C081127) respectively. The concentration of CBD-RGD protein at different concentrations of 1〇 and 20 mg/g is used as the experimental group, and is not included in the CBD- The RGD protein was a control group. Table 1. Experimental group of different concentrations of CBD-RGD protein in the composition coated with cells coated with cell composition CBD-RGD protein (mg/g) hBMSCs hPDMCs alginate nCDHA ( Adsorbed with TGF-p3) Control group 0 + + + Experimental group 10 + + + 20 + + + Firstly, human bone marrow mesenchymal stem cells (hBMSCs) with a cell density of 1 4〇6 cells/ml and human placental mesenchymal stem cells ( hPDMCs were mixed with 10 mg/g and 20 mg/g of CBD-RGD protein, respectively, and then added with adsorbed TGF-β; (1 pg/100 ml) of nCDHA, the mixture (containing stem cells, CBD-RGD and The alginate solution was further added to the nCDHA adsorbed with TGF-p3, and finally the mixture was placed in a sterile syringe. A suitable amount of 1 〇 2 mM calcium chloride solution was added to a 6-well culture dish, and the cells were dropped into a 6-well culture dish with a 29-gauge needle to form alginate microbeads having a diameter of about 2 mm ( Alginate bead), let stand for 1 minute, after the gel is completely removed, carefully remove the calcium chloride solution with a plastic dropper, and add a proper amount of 0.15M sodium vapor solution to wash the 14 i 201136622 alginate microbeads. The medium (Ch〇ndr〇genic coffee (4) was placed in a 37C 5/〇C02i mouthbox for 21 days, and the culture was changed every three days. After 21 days of culture, the alginate beads were washed with PBS. Then, the sample was immersed in the 55-nose drop soda solution for 10 minutes. After the brown algae win beads were completely dissolved, the cells were collected by centrifugation, and the content of glucosamine-polymerized sugar (GAG) was analyzed, and the indigo blue staining and the su Wood violet-eosin staining. The results are shown in the fourth, fifth and sixth figures. Please refer to the fourth figure, which is used in the repair of cartilage tissue to combine different / minute CBD - Glucosamine polymeric sugar coated with a composition of cells of the RGD protein (GA) G) Analysis of the results of the content analysis. It can be observed from the figure that since the CBD_RGD protein can activate cells and promote φ into cell proliferation, thereby secreting a larger amount of extracellular matrix, glucosamine polymeric sugar (GAG) will be placed along with the CBD. -RGD protein concentration increased, but when the CBD-RGD protein concentration was 10 mg/g, there was a significant difference in extracellular matrix secreted by human bone marrow mesenchymal stem cells (hBMSCs) and human placental mesenchymal stem cells (hPDMCs). (p &lt; 0.001) 凊 See also the fifth and sixth figures, wherein the fifth figure is a coated cell with a different concentration of CBD-RGD protein in the composite material for repairing cartilage tissue of the embodiment of the present invention. The results of the indigo blue staining of the composition are shown in the sixth embodiment of the present invention. The hematoxylin-eosin containing the cell-bound composition of different concentrations of CBD-RGD protein in the composite material for repairing cartilage tissue. Staining results. Human alveolar stem cells (hBMSCs) were observed in the tissue sections of Aldrin blue staining without CBD-RGD protein and two different concentrations of CBD-RGD protein. There was no significant difference in cartilage-induced differentiation and no lacunae formation; and in the results of hematoxylin-eosin staining, only when the concentration of CBD-RGD protein was 1 〇mg/g, it was observed. The phenomenon of cell aggregation to black dots, which is a sign that cells tend to mature into cartilage. On the other hand, human placental mesenchymal stem cells (hPDMCs) induce cartilage-induced alfalfa staining and hematoxylin-eosin staining results. When there is no CBD-RGD protein, dimples can also be formed, but when the concentration of CBD-RGD protein is 1〇mg/g, the dimples are widely distributed, complete and more numerous. These results indicate that human placental mesenchymal stem cells (hPDMCs) are more likely to induce chondrogenesis than human bone marrow mesenchymal stem cells (hBMSCs) in a three-dimensional alginate culture, IS1 15 201136622 and when CBD-RGD protein concentration At 10 mg/g, the effect of inducing stem cells for chondrogenesis is optimal. Further, the embodiment of the present invention is for repairing a cartilage tissue containing only human human placental mesenchymal stem cells, TGF-β3 and alginate-coated cell-forming composition by the aforementioned injection method, thereby achieving the effect of repairing cartilage. Example 3 Preparation of Composite Material for Repairing Cartilage Tissue First, a poly lactic-co-glycolic acid (hereinafter referred to as PLGA) precision scaffold was prepared, and 20 g of PLGA (50) was prepared. /50,Kyoto) is dissolved in 80 g of l,4-dioxane (Tedia) to prepare 20% PLGA, followed by liquid-frozen deposition manufacturing (LFDM) to make 4 angles (0°) /45°/90°/135°) precision bracket (see also figures 7a and 7b), the precision bracket has a diameter of 6.5 mm and a thickness of 2.5 mm, and its fiber diameter (Φη aperture, Φη ) is 0.2 mm, and the interfiber spacing adjacent fibers (dh) is 0.7 mm, and the microstructure of the precision scaffold is as shown in the scanning electron microscope (SEM) images of Figs. 8A and 8B. In addition, this precision scaffold has the advantages of high porosity, high specific surface area, complete permeability between pores, controlled porosity and independent pore size control. Then, a cell-containing composition containing stem cells, CBD-RGD protein, nCDHA adsorbed with TGF-p3, and brown algae is added to a PLGA precision scaffold to prepare a composite material for repairing cartilage tissue according to an embodiment of the invention. . The effect of the composite material for repairing cartilage tissue on the induction of chondrogenesis in different culture days is to find the appropriate number of stem cell-induced culture days, and the present invention analyzes the composite material for repairing cartilage tissue in different cultures. The effect of days on stem cell-induced chondrogenesis. Please refer to Table 2, which will contain only human placental mesenchymal stem cells (C080520), human placental mesenchymal stem cells (C080520) and alginate, and human placental mesenchymal stem cells (c〇8〇52〇), alginate, CBD- RGD protein and nCDHA adsorbed TGF-β3, respectively, were combined with pLGA precision scaffolds for a culture time of 1, 7, 21 and 36 days. IS1 201136622 Table - Experimental group for repairing cartilage shouting composites in different culture days of female lining guide cartilage formation PLGA precision scaffold human placental mesenchymal stem cells (C080520) Alginate has adsorbed TGF-p3 nCDHA CBD -RGD protein experimental group A + one - experimental group B + + one experimental group C + + + + experimental group A will be cultured to the sixth generation of human placental mesenchymal stem cells (hPDMCs) as described in Example 1 DMEM low glucose culture The solution is prepared into a cell fluid with a cell concentration of 1 x 106 cells/scaffoid (that is, a PLGA precision scaffold containing 1 X 1 6 human placental mesenchymal stem cells), and 3 〇... cytosol (about 1 X 106 human placenta) Mesenchymal stem cells were dropped into a PLGA precision scaffold and placed in a 37 ° C, 5% C〇2 incubator for 2 hours to attach the cells to the scaffold, and then the cell and scaffold mixture was transferred to a 12-well culture plate. In the experimental group B, the human placental mesenchymal stem cells (hPDMCs) cultured to the fifth generation were prepared into a cell concentration of 1×106 cells/scaffold with a 1.2% alginate solution, and 30 μΐ of the cell liquid (about 1). xlO6 human placental mesenchymal stem cells were dropped into the PLGA precision scaffold, the cell fluid was filled in the entire scaffold, and the prepared cell and scaffold mixture was placed in a 6.5 mm transfer plate (transwell, Coming) at 102 mM calcium chloride. The solution was immersed for about 10 minutes. After the gel was added, the solution was washed with an appropriate amount of sodium chloride solution, washed with a culture solution, and the mixture of the cells and the scaffold was transferred to a 12-well culture dish. In the experimental group C, human placental mesenchymal stem cells with a cell concentration of 1×106 cells/scaffold were mixed with a concentration of 10 mg/g of CBD-RGD protein (ie, 1〇mg CBD-RGD/g alginate), and then added. The tCD-p3 nCDHA has been adsorbed (the NGFHA at 500 ppm is added to the TGF-p3 concentration of 〇.〇1 pg/ml, and the mixture is placed in a 37 ° C, 5% CO 2 incubator for 1 hour. Then, add 1.2% alginate solution to form a cell mixture (that is, a composition coated with cells), and mix 30 μΐ of the cells into a PLGA precision scaffold to fill the cell mixture. 17 201136622 Full of the stent, the prepared cell and stent mixture was placed in a 6.5 mm transwell and soaked in a 102 mM chlorinated mother solution for about 1 minute. After the gel was added, an appropriate amount of sodium chloride was added. The bath was washed, washed with the culture solution, and the cell and scaffold mixture was transferred to a 12-well culture dish. Experimental groups A, B and C were cultured for 1 day, 7 days, 21 days '36 days, respectively, in a cartilage differentiation medium containing 1 ng/ml of TGF-p3 growth factor as described in Example 1, and induced during differentiation. The culture solution was changed every 3 days, and in addition, each group was repeated three times (n=3). At the end of the induction differentiation time, the scaffold was washed twice with PBS, and after cold-dried, cell number analysis, glucosamine-polymerized sugar (GAG) content analysis, type II collagen content analysis, and hematoxylin φ red staining analysis, Alcian blue staining analysis, and immunofluorescence staining analysis of type 2 collagen. The results are shown in Figures 9 through 14. Please refer to the ninth figure, which is a graph showing the number of chondrocyte production of the composite material for repairing cartilage tissue after 1, 7, 21 and 36 days of culture in the embodiment of the present invention, as shown in the figure, on the first day of culture. In the composite material for repairing cartilage tissue, the amount of cells in the composite material for repairing cartilage tissue is about 60 to 70%, and the number of cells in each experimental group increases as the culture time increases. Since the experimental group has alginate, the cells can be wrapped in a precision scaffold, so the number of cells growing increases with the incubation time, while the experimental group A does not have alginate, so most of the cells will be lost with the replacement of the culture solution. Therefore, the experimental group A was cultured • The number of cells on the 7th day showed a downward trend. On the other hand, at 7 days and 21 days of culture, the number of cells in the C line of the experimental group A and B' had the largest number of cells, and the number of cells was significantly different from that of the experimental group. Please refer to the tenth figure. This figure is a graph showing the analysis results of the content of glucosamine polymerized sugar (GAG) in the composite material for repairing cartilage tissue after 7, 2 and 36 days of culture in the embodiment of the present invention. On days 7 and 2i, the content of glucosamine polymerization (gag) in the extracellular matrix secreted by c cells in the experimental group was significantly higher than that in the experimental group A and Β (ρ&lt;0.05) and was cultured to the next day. During the period, the content of glucosamine-polymerized sugar (GAG) in each experimental group increased with the increase of culture time, while at the 36th day of culture, the content of glucosamine-polymerized sugar (GAG) in each experimental group decreased a lot. However, the glucosamine polymerization (GAG) content of the experimental group C is still the highest in the actual 201136622. Please refer to the tenth-figure, which is a composite material for repairing cartilage tissue in the embodiment of the present invention. The results of the analysis of the type II collagen content after 21 and 36 days showed that, on the 7th day and the 21st day after the differentiation culture, the experimental group had no side-to-second type collagen expression, compared with In the experimental group A, the experimental group (10) measured the second type of glue Protein expression, and the expression levels on day 7 ^ may be about __ days, 36 days Peihe secret, the performance of the second type collagen in an amount of two experiments ^ butterfly significantly reduced (ρ &lt; 0.001).

Referring to the twelfth figure, the figure is a composite material for repairing cartilage tissue in the embodiment of the present invention, in the scale 7 and _6 day-like Su-like red staining and entanglement diagram, by the cleaning tissue in the precision branch = internal growth situation The results showed that 'experiment group VIII only observed the presence of precision scaffolds on the 7th and 21st day of culture, and no _ to cell expression, cell expression was observed after the 36th day of culture' experiment group Β and c culture After the 7th, and after 36 days, the performance of the cells attached to the precision scaffolds was observed (4), and the phenomenon of 骨 _ (4) was also observed. The experimental red color was obvious when the culture was on the first day of culture. The darker color of the dimples. See the thirteenth circle. This figure is a graph of the results of the analysis of the indigo blue staining of the composite material for repairing cartilage tissue in the embodiment of the present invention after 7, 21, and 36 days of culture. The results of the figure show that the hybrid culture has no 纟 纟 基质 基质 _ _ 21 21 21 , , _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Performance situation, and there is a clear and dark acupuncture) 'and there is a towel Table cells of blue dots appear. ° : Refer to the fourteenth figure 'Description of the invention _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Glue = dense branch &amp; surrounding 'and the second type of collagen 2 is less distributed between the fiber and the fiber of the precision scaffold, and the second Nana County white m-money gamma after m36 days, by the picture... In the 7th and 21st days, the experimental group had the performance of sputum type II collagen, and the experiment was the same as the ninth to eleventh pictures. In the experimental group, the number of cells reached the highest at 201136622 on the 7th day of culture and lasted for 21 days. And its glucosamine-polymerized sugar (GAG) system reached the highest value on the 21st day of culture'. This indicates that the experimental group C has the effect of promoting the differentiation of stem cells to form chondrocytes early and the chondrogenesis period is between 7 and 21 days. Since nCDHA adsorbed with TGF-p3 in experimental group c can release TGF-h through a diffusion mechanism, direct differentiation of stem cells can be achieved, thereby avoiding uneven differentiation; the number of cells in experimental group B and glucosamine-polymerized sugar (GAG) The content reached the highest value on the 21st day of culture, which represents The experimental group B cell growth and differentiation at the same time, but in general, growth and differentiation of the cells should be performed in different periods, so that the experimental group B unevenness differentiations. Please also refer to the twelfth to fourteenth, experimental group C has cell clusters (cellclusters) and has a trap (lacunae) for the cartilage maturation, there is no uneven differentiation, and experimental group B is φ The distribution of the extracellular matrix away from the precision scaffold fibers is more complete and distinctly differentiated than the extracellular matrix of the precision scaffold fibers. In addition, these results show that human placental mesenchymal stem cells undergo a cartilage stromal secretion period from day 7 to day 21 after differentiation induced by cartilage differentiation medium. After 21 days, cartilage matrix secretion tends to mature cartilage, so Jiazhi induced differentiation and culture time was 21 days. On the other hand, from these results, it was found that although experimental group B did not add nCDHA and CBD-RGD, it still induced chondrocyte differentiation and promoted secretion of extracellular matrix, while experimental group c was added by nCDHA and CBD-RGD. It has a better differentiation effect. The description includes the human fetal placental mesenchymal stem cells, the TGF-β3 and the PLGA precision scaffold, and the composite φ material for repairing the cartilage tissue of the embodiment of the invention can achieve the effect of inducing chondrocyte differentiation and repairing the damaged cartilage tissue, and The addition of nCDHA and CBD-RGD can further promote the repair effect. Example 5 Effect of Different Stem Cell Species on the Induction of Chondrogenesis in a Composite Material for Repairing Cartilage Tissues The present inventors further analyzed the effects of different types of stem cells in the composite material for repairing cartilage tissue on stem cell-induced chondrogenesis. Please refer to Table 3, experimental groups A, B and c all contain stem cells, alginate, CBD-RGD protein 'NSDHA that has adsorbed TGF-β3, and PLGA essence, branch's stem cells of the test group A are human bone marrow The stem cells of the stem cells (hsc_i) and the experimental group B were human bone marrow mesenchymal stem cells (C1008), and the stem cells of the experimental group c were human placental mesenchymal stem cells (C081127).

I 20 201136622 Table 3. Effects of different stem cell types on the induction of chondrogenesis in composites used to repair cartilage tissue PLGA precision scaffold stem cells alginate has adsorbed TGF-p3 nCDHA CBD-RGD protein experimental group A human bone marrow mesenchymal stem cells ( HSC-1) + + + experimental group B human bone marrow mesenchymal stem cells (C1008, + + + experimental group C human placental mesenchymal stem cells (C0811271 + + + experimental group A, B and C respectively have a cell concentration of 1 X 106 cells) /scaffold human bone marrow mesenchymal stem cells (HSC-1), human bone marrow mesenchymal stem cells (C1008) and human placental mesenchymal stem cells _ (C081127) mixed with CD-RGD protein at a concentration of 10 mg/g, added to the adsorbed nCDHA of TGF-p3 (take appropriate amount of nCDHA and add TGF-p3 at a concentration of 0.01 gg/ml, mix evenly, place the mixture in 37 &lt; t, 5% CO 2 incubator for 1 hour), then add 1.2% alginate solution to form a cell mixture, 3 〇μΐ cells were mixed into a PLGA precision scaffold, the cell mixture was filled with the entire scaffold, and the prepared cell and scaffold mixture was placed in a 6.5 mm transfer. Disk (tr Answell) and soaked in 1〇2 mM calcium carbonate solution for about 1 minute, after gelation, add appropriate amount of sodium vapor solution to clean, then 'wash with culture solution' and then move the cell and scaffold mixture to 12 wells. In the culture tray, the culture medium was cultured for 21 days in the cartilage differentiation medium containing 1 ng/mi of TGF_p3 growth factor as described in Example 并, and the culture solution was changed every 3 days during the differentiation induction. In addition, the Φ group was examined. The system was repeated three times (n=3). After the end of the differentiation time, the scaffold was washed twice with pBS and lyophilized, the cell number analysis, the content of glucosamine polymerized sugar (GAG), and the second type collagen were performed. Analysis of protein content, analysis of hematoxylin-eosin staining, analysis of indigo blue staining, and immunofluorescence staining of type II collagen. The results are shown in Figures 15 to 18. Please also refer to the tenth. Figure 5 to 17 is a graph showing the number of chondrocytes produced by different stem cell types in the composite material for repairing cartilage tissue in the composite material for repairing cartilage tissue, and the content of glucosamine polymeric sugar (GAG). The results of the results and the analysis of the results of the second type of collagen analysis are shown by the results of δ Xuan. After 21 days of culture, the number of cells grown in the experimental group (Ρ&lt;〇_〇5) and the extracellular matrix glucosamine polymerization. Sugar (GAG) content &amp;&lt;〇〇〇1) are compared with experimental group a 21 201136622

The experimental group c was significantly more than the experimental groups A and B, especially the glucosamine polymeric sugar (GAG) content was about twice as high as B.

Please refer to the eighteenth figure, which is a method for repairing cartilage tissue in the composite of cartilage tissue, which is used for repairing the surface of cartilage, and the hematoxylin-eosin staining analysis, the indigo blue analysis and the second division white immunization. The results of fluorescence staining analysis showed that the results of Glucosamine Polymeric Glucose (GAG) and Type II Collagen were observed in Groups A, B and C, and most of the Glucosamine Glycans were tested. Mosquito _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ There is a gap in the duck on the edge, and the distribution of glucosamine polymerization (GAG) and the second type of prion protein in the experimental group C is more complete and the dark blue cell mass in the extracellular matrix stained blue Small dots are more obvious. On the other hand, although the experimental groups A and B are the same as the human bone marrow mesenchymal stems, 〇M, ci〇〇8), but the performance of the second nanoprotein is poor, this represents the human leaf stem cells. The amount of extracellular matrix secretion varies with the individual from the stem cell source, and the second type collagen expression from human face lateral stem cells (〇127127, CG8G52G) from different individuals is compared. After 21 days of induction, The expression of the second type of collagen is about 6 (four) scaff〇id, there is no significant difference (please also refer to the tenth-figure and the seventeenth figure), in addition, after 21 days of induction, human fetal system from different individuals Leaf stem cells (CG8丨丨27, C_52G) (iv) Schiffin-polymerized vinegar (GAG) showed s of about 80 pg/scaffold (please also refer to the tenth and sixteenth figures), which shows human placental mesenchymal stem cells. The source individual does not affect the amount of extracellular matrix secreted. Therefore, these results show that the human placental mesenchymal stem cell line used in the composite material for repairing cartilage tissue of the present invention has better ability to induce differentiation and is more stable and not due to stem cells than human bone marrow mesenchymal stem cells. The difference in the source of the individual affects its ability to induce differentiation 0. BRIEF DESCRIPTION OF THE DRAWINGS The first figure is a flow cytometric analysis of cell surface proteins of stem cells in a composite material for repairing cartilage tissue according to an embodiment of the present invention. In the figure, ~(e) is a human mesenchymal stem cell, (f) ~ 〇) 22 201136622 is a human placental mesenchymal stem cell. The eighth figure is a diagram of the human fetal cells in the composite material for repairing cartilage tissue according to the embodiment of the present invention, and the result of the analysis of the differentiation of chondrocytes by the cartilage differentiation agent containing the transforming growth factor (9) into the chondrocyte. The magnification is i(8) times, E: extracellular matric, L: lacuna. The second figure is the human placenta cells in the composite material for repairing cartilage tissue in the embodiment of the invention, for reading growth The results of the analysis of the other-indigo blue staining induced by factor (10) in the cartilage differentiation agent. The magnification in the figure is double, E: extracellular matric, L· : lacuna. • The third embodiment of the present invention is used for repairing the composite of human chondrocytes in the composite of cartilage tissue, and the second type collagen egg which is induced to differentiate into chondrocytes by transforming growth factor (medicine-(9) cartilage differentiation agent) Immunofluorescence staining analysis results. The magnification in the figure is the reward, and the arrow points to the second type of collagen. The third picture of the county invention _ 麟修录骨_ _ 磐 人 人 娜 娜 娜 娜The results of immunostaining staining analysis of the other-type-type collagen egg which is induced to differentiate into chondrocytes by transforming growth factor (TG_-based cartilage differentiation agent), the magnification is 2〇〇, the arrow points to The second type of collagen is a graph showing the results of analysis of glucosamine polymerized sugar (GAG) content of the cell-coated composition of the cartilage tissue combined with different concentrations of cbd_rgd protein in the cartilage tissue. Representative p&lt;0.05, 氺氺氺 represents; ?&lt;〇.〇〇ι. The fifth figure is a coated cell containing a different concentration of CBD-RGD protein in a composite material for repairing cartilage tissue according to an embodiment of the present invention. The results of the indigo blue staining of the composition. In the figure, (a) to (c) are sequentially stained with human bone marrow mesenchymal stem cells bound with C, 1〇, 20 mg/g of CBD-RGD protein, (d) ~(f) is the result of human placental mesenchymal stem cell staining of cbd-RGD protein combined with 〇, 1〇, 2〇mg/g. The magnification is 2〇〇, c: cell, E : extracellular matric, L: iacuna 〇 sixth image Embodying Example composites for the repair of cartilage tissue in combination with different coating concentrations of the protein CBD-RGD has _ hematoxylin eosin staining of cells in the composition of FIG. FIG.

ISJ 23 201136622

Medium (a) to (c) are sequentially stained with human bone marrow mesenchymal stem cells bound with 0, 10, 20 mg/g of CBD-RGD protein, and (d) to (f) are sequentially bound to sputum, Human placental mesenchymal stem cell staining results of 1〇, 2〇mg/g CBD RGD protein. Magnification is 2〇〇, E: extracellular matric 'L: Iacuna. The seventh embodiment is a structure of the composite material for repairing cartilage tissue of the embodiment of the present invention. The structure of the eight-precision stent is not intended. The fifth embodiment is a composite material for repairing cartilage tissue in the embodiment of the present invention. A schematic view of another structure of the stent. Fig. 2 is a front view of a scanning electron microscope (SEM) of a pLGA precision stent in a composite material for repairing cartilage tissue according to an embodiment of the present invention. Scanning electron microscope (SEM) cross-sectional view of the pLGA precision scaffold in the composite of repaired cartilage tissue. The ninth figure is the cartilage of the composite material for repairing cartilage tissue in the cultured sputum, 7, 21 and 36 days after the invention is used. A graph showing the number of cells produced. In the figure, * represents p &lt; 〇〇 5, and wood ^ represents ^ 0 · 0 (Η. The tenth figure is the implementation of the present invention. The composite material for repairing cartilage tissue is cultured. A graph showing the results of analysis of glucosamine polymerized sugar (GAG) content. In the figure, * represents ρ &lt; 〇〇 5, *** represents; ? &lt; 0. 瘫 Tenth - Figure is an embodiment of the present invention for repairing cartilage tissue Composite materials in culture 7, Μ and % A graph of the results of analysis of the type II collagen content of the day. In the figure, * represents *** represents ρ &lt; 0·001. The twelfth figure is a composite material for repairing cartilage tissue in the embodiment of the present invention in culture 7, 21 And the results of the analysis of Sumu purple and eosin staining after 36 days. The tissues (a) to (e) are the staining results of the experimental group A, Β and C on the 7th day of maintenance, (d) ~ ( f) The staining results of the experimental groups a, β and C in the second day of culture were sequentially followed, and (gHi) was sequentially the $9 color result of the experimental group a and B' spot c on the 36th day of culture. The magnification was 100. L, L: lacuna, s: plga precision scaffold 2 thirteenth figure is a composite material used in the repair of cartilage tissue in the embodiment of the present invention. Results. Fig. (8) ~ (6) in order to cultivate the magical reality: 201136622 ^ color results, _ in order to train f 2i days of real loss, feet, · · · fruit, (g) ~ (1) in order to train On the 36th day, the experimental group A, the wood color is the Qing times, the L color is the height of the na, the S: PLGA precision color, and the fruit. The magnification is used to repair the composite material of the cartilage tissue in the culture 7, 21 and 36 days The results of immunofluorescence staining analysis of the original protein. In the figure, (aHe) is the staining result of the test group A, B and C for 7 days, and (d) ~ (f) sequence '. On the 21st day, the experimental group A, 'wood, and (g) ~ (1) were sequentially subjected to the experimental group A and B^c on the 36th day of culture. The magnification was doubled, S: PLGA precision scaffold, τ: The second type of collagen color knot is the fifth figure of the present invention, which is a result of the number of different stem cells generated by the composite material for repairing cartilage tissue. In the figure, * represents the corpse &lt; No.: Liutu County, Mouth, Guanlin repair, bone tissue composite material domain analysis of glucosamine polymer sugar (gag) content analysis. In the figure, *** represents the household &lt;甫 The seventeenth figure is the result of the analysis of the type II collagen content of the composite domain domain cells of the pure soft woven fabric. In the figure, 氺 represents P &lt; 0.05. Eighteenth is a group of different stem cells in a composite material for repairing cartilage tissue according to an embodiment of the present invention

Weaving slice staining analysis results. In the figure, (a) to (c) are human bone marrow mesenchymal stem cells (the analysis of the Su-Purple-Eosin staining of the ship U, the staining analysis of Al and the results of the second sputum protein immuno-light staining analysis, (dH 〇 Sequence of human bone marrow mesenchymal stem cells (cl〇〇8)

Staining analysis, Alcian blue staining analysis and type II collagen immunofluorescence staining analysis of Z fruit, (g) ~ (i) sequentially for human placental mesenchymal stem cells (C081127) Sumu purple · Ery Red: Analysis, Arnus staining analysis and second-type collagen immunofluorescence color analysis results: the double rate is 100 times, L: lacuna, S: PLGA precision stent, τ: type II collagen. 〃 [Explanation of main component symbols] ΪΓ1 25 201136622 Sequence Listing &lt;110&gt;National Health Research Institute&lt;120&gt; Composite material for repairing cartilage tissue and preparation method thereof &lt;130> 990167-11 &lt;160〉 1 &lt ;170&gt; Patentln version 3.3 &lt;210&gt; 1 φ &lt;211&gt; 48 &lt;212&gt; PRT &lt;213&gt; Synthetic &lt;220&gt;&lt;223&gt; Cellulose-binding functional region-RGD attachment sequence (CBD-RGD) ) &lt;400〉 1

Pro Thr Gin His Trp Gly Gin Cys Gly Gly lie Gly Tyr Ser Gly Pro 15 10 15

Thr Val Cys Ala Ser Gly Thr Thr Cys Gin Val Leu Asn Pro Tyr Tyr 20 25 30

Ser Gin Cys Leu Pro Thr Thr Pro Thr Gly Arg Gly Asp Ser Ala Ser 35 40 45

Claims (1)

201136622 VII. Patent application scope: 1. A composite material for repairing cartilage tissue, comprising: a cell-coated composition comprising human placenta-derived mesenchymal cells and a alginate (alginate); wherein the cell-coated composition induces differentiation of the human placental mesenchymal stem cells into chondrocytes by a transforming growth factor-β. 2. The composite material for repairing cartilage tissue according to claim 1, further comprising a polylactic acid-polyglycolic acid (PLGA) precision scaffold. 3. The composite material for repairing cartilage tissue according to claim 2 or 2, wherein the cell-coated composition further comprises an RGD (Arginine-Glycine-Aspartate) attachment sequence. The composite material for repairing cartilage tissue according to claim 3, wherein the peptide having the RGD attachment sequence is as shown in SEQ ID NO: 1. Cellulose binding functional region of amino acid sequence · RGD attachment sequence (CBD-RGD). 5. The composite material for repairing cartilage tissue according to claim 4, wherein the concentration of the cellulose-binding functional region·RDD attachment sequence is about 1 mg/g. The composite material for repairing cartilage tissue according to the invention of claim 2, wherein the transforming growth factor-β system is contained in the cell-coated composition. 7. The composite material for repairing cartilage tissue according to claim 1, wherein the cell-coated composition further comprises a biomedical ceramic particle. 8. The composite material for repairing cartilage tissue according to claim 7, wherein the biomedical ceramic particle system adsorbs the transforming growth factor_β e 9 as described in claim 7 or 8. The composite material for repairing cartilage tissue, wherein the biomedical medicinal granule is nanometer-deficient hydroxyapatite (nan〇_caldum sulphur (10) hydroxyapatite). 201136622 10, as described in item 9 of the towel 4 patent for repairing cartilage tissue ▲ nano-calcium-deficient calcium-hydroxyapatite concentration of about 50-1000 ppm. The composite material according to any one of claims 1, 2, 6 or 8, wherein the transforming growth factor-β is a transforming growth factor. ^ Weaving 12, as claimed in the patent scope of item u for repairing cartilage tissue composites, the concentration of transforming growth factor-β3 is about 0 〇 1 pg / mh /,. Λ 13. A composite material for repairing cartilage tissue, as described in Patent No. i or 2, wherein the concentration of the human placental mesenchymal stem cells is about 〇1〇6 cells/cells/scaffold. , 14. For example, please refer to the composite of soft-woven fabrics as described in item i or item 2, wherein the concentration of the alginate is about 1.2%. 15. The composite material for repairing cartilage tissue according to claim 2, which can induce about 6 Mg/scaffold type II collagen and about 8 〇μΕ/scaffold glucose after 21 days of differentiation. Amine polymerized sugar. 16. A method of preparing a composite material for repairing cartilage tissue, the method comprising: rinsing human placenta-derived mesenchymal cells and alginate to form a coated cell The composition; and the cell-coated composition induces differentiation of the human placental mesenchymal stem cells into chondrocytes by a transforming growth factor-β. 17. The method of claim 16, wherein the step further comprises combining the cell-coated composition with a polylactic acid-polyglycolic acid (PLGA) precision scaffold and then inducing Differentiation. 18. The method of claim 16, wherein the human placental mesenchymal stem cell line is first coupled to a peptide having an RGD (Arginine-Glycine-Aspartate) attachment sequence (RGD-containing peptide). )mixing. Γ The method of claim 18, wherein the peptide having the RGD-attached sequence is a cellulose-binding functional region having an amino acid sequence as shown in SEQ ID N:: _ RGD Attach the sequence (CBD_RGD). 20. The method of claim 19, wherein the concentration of the cellulose binding functional region _ RGD attachment sequence is about 10 mg/g. 21. The method of claim 16, wherein the human placental mesenchymal stem cell line is mixed with the transforming growth factor _β and then added to the alginate. 22. The method of claim 16, wherein the cell-coated composition further comprises a biomedical ceramic particle. 23. The method of claim 22, wherein the biomedical Taojing particle system adsorbs the transforming growth factor-β. 24, too as a towel, please specialize in the method described in Dijon or Μ, the towel of the doctor called the granules of nano-calcium deficient hydiOxyapatite (nan〇-calcium deficient hydiOxyapatite). 25. The method of claim 5, wherein the concentration of the nano-enriched oxyapatite is about 50-1000 ppm. The method of claim 16, wherein the transforming growth factor-β is transforming growth factor-β3. </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The agronomic degree of human mesenchymal stem cells is 1 X 1〇6 cells/scaffold. The method of claim 16 or 17, wherein the alginate concentration is 丄2%.