Preparation and application of improved composite microspheres of cadherin fusion protein

Abstract

Preparation and application of improved composite microspheres of cadherin fusion protein. The present invention relates to modified matrices of E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein and preparation methods thereof, as well as applications thereof in promoting cellular immune regulation function and / or promoting cartilage repair.

Description

Technical Field

[0001] The present invention relates to the preparation of modified biomaterial composite microspheres and the biomedical basis and application development fields of regulating the maintenance of stem cell functions, especially the research and development of stem cell immune regulation and cartilage repair fields. More specifically, it relates to the preparation of biomaterial shell-core structure composite microspheres, and to the synergy of epithelial cell cadherin (hE-cadherin)-Fc fusion protein and / or neural cell cadherin (hN-cadherin)-Fc fusion protein and the new use of modified biomaterial composite microspheres, which promotes mesenchymal stem cell immune regulation and cartilage repair function. Background Art

[0002] Mesenchymal stem cells (MSCs) have become the main candidate drugs for cell therapy and tissue repair due to their wide range of sources, self-renewal and paracrine abilities. Combining MSCs with microspheres to prepare engineered cells can better preserve the innate properties of MSCs and enhance their therapeutic effects. The uniformity of the microsphere particle size is crucial to the homogeneity of the internal structure of the engineered cells and the release of the soluble proteins and small molecules they carry.

[0003] Traditional microsphere preparation technology is mainly emulsion polymerization, which relies on different shear forces caused by different rotation speeds generated by magnetic stirring or ultrasound to prepare microspheres. The uneven particle size and batch-to-batch differences of the produced microspheres can easily lead to inconsistent degradation of the microspheres and decreased stability of drug release. Microfluidic droplet technology uses stable shear forces between immiscible fluids to not only effectively prepare highly uniform monodisperse microspheres without secondary purification, but also can prepare microspheres of different structures by adjusting the microfluidic chip structure, device and microfluidic process. However, its chip processing cost is high, the process is complex and not easy to operate.

[0004] Using biomaterials to construct extracellular microenvironments to guide cell behavior and function plays an important role in modern regenerative medicine and tissue engineering. Current research generally believes that the immune regulation ability of MSCs is plastic, that is, the immune regulation ability of MSCs can be acquired or enhanced during in vitro culture. Therefore, how to obtain stem cells with high biological functions by artificially regulating the interaction between cells is a key issue that researchers have been working on. Cadherin (abbreviated as cad in this article) is a type of Ca 2+E-cadherin is a transmembrane glycoprotein that is dependent on the cell surface and is an important member of the cell adhesion molecule family. Classical cadherin is a class of transmembrane glycoproteins with strict cell specificity. Its extracellular domain mediates homologous cell connections (i.e., adhesion connections), and its intracellular domain is connected to intracellular α, β, and γ-catenin, regulating actin fibers and participating in a variety of signal transductions. It has the functions of establishing cell polarity, maintaining cell morphology and tissue and organ integrity, and is also an important regulatory factor for cell differentiation and morphogenesis. E-cadherin (E-cad) is a class of transmembrane glycoproteins that mediate cell-to-cell adhesion in epithelial cells and embryonic stem cells. It is essential for epithelial formation, cell rearrangement, tissue morphogenesis, establishment of cell polarity, and maintenance of tissue structure in early mouse embryos. N-cadherin protein (N-cad) is one of the important markers of the interstitial condensation process during cartilage morphogenesis and development. High expression of N-cadherin has been shown to promote the interstitial condensation process of mesenchymal stem cells, but continued enhancement of N-cadherin expression will also inhibit the subsequent cartilage differentiation process of interstitial condensation, so the temporal and spatial regulation of the expression of N-cadherin in cells is crucial. In addition, it is difficult for chondrocytes differentiated through directional induction to maintain the characteristics of stable hyaline cartilage, and hypertrophy and other phenomena are very likely to occur, and then calcification occurs, thereby losing the ability and physiological function of chondrocyte-specific matrix synthesis.

[0005] There are many fusion proteins based on the IgG Fc segment and the extracellular domain of cadherin that have been applied in the research of tissue engineering and regenerative medicine. For example, E-Cad-Fc and N-Cad-Fc are used as one of the modified components of the extracellular matrix to study the effects on the regulation of cell behavior.

[0006] However, so far, there is no report in the prior art on the promotion of the immunoregulatory function of mesenchymal stem cells by human E-cadherin-Fc fusion protein and human neural cadherin-Fc. The research on E-cadherin is more focused on promoting epithelial cell function, and there is no research on promoting the differentiation of stem cells into chondrocytes by combining human E-cadherin-Fc fusion protein and human neural cadherin-Fc, inhibiting the calcification of mature chondrocytes by regulating related signal pathways, and repairing articular cartilage. Summary of the invention

[0007] The inventors have found that the use of core-shell composite microspheres (such as PLGA, PCL, chitosan, heparin, hyaluronic acid, collagen) and the modification with E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein will be beneficial to the application of mesenchymal stem cell immunoregulation and cartilage repair.

[0008] For example, by using functionalized modified composite microspheres of E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein, the dual advantages of fusion protein and three-dimensional culture can be combined, and the matrix with fusion protein fixed on the surface can be introduced into the interior of stem cell aggregates at the three-dimensional level. The paracrine and immune regulatory capabilities of stem cells on the matrix surface are regulated through the binding of cell surface cadherin with the same type of cadherin-Fc on the matrix surface, further coordinating exogenous cellular inflammatory factors, biomimetically constructing an extracellular microenvironment, and regulating the immune regulatory function, cell differentiation and cartilage repair function of stem cells.

[0009] One aspect of the present invention relates to the use of E-cadherin-Fc fusion protein and N-cadherin-Fc for culturing stem cells or promoting the immunoregulatory function of mesenchymal stem cells. Preferably, the E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein are connected to a matrix.

[0010] Another aspect of the present invention relates to the use of E-cadherin-Fc fusion protein and N-cadherin-Fc in chondrocyte differentiation, inhibition of mature chondrocyte calcification and repair of joint defects.

[0011] On the other hand, the present invention relates to the preparation of a modified matrix, comprising modifying the matrix with E-cadherin-Fc fusion protein and neural cell cadherin-Fc, wherein the matrix is ​​a microsphere, preferably the microsphere particle size is 10-50 microns (preferably 20±5 microns), preferably the surface hydrophobic microsphere, and more preferably the microsphere is selected from PLGA / chitosan-heparin core-shell structure composite microspheres.

[0012] In the specific embodiments of the various aspects of the present invention, the E-cadherin is human E-cadherin; the N-cadherin is human N-cadherin; and the Fc is the Fc of human IgG (preferably IgG1).

[0013] In the specific embodiments of the various aspects of the present invention described above, the sequence of the E-cadherin is shown by SEQ ID NO: 2, and the sequence of Fc is represented by the sequence of SEQ ID NO: 3; preferably, the sequence of the E-cadherin-Fc fusion protein is represented by the sequence of SEQ ID NO: 1; the sequence of the N-cadherin is shown by SEQ ID NO: 5, and the sequence of Fc is represented by the sequence of SEQ ID NO: 3; preferably, the sequence of the N-cadherin-Fc fusion protein is represented by the sequence of SEQ ID NO: 4.

[0014] In specific embodiments of the various aspects of the present invention described above, the hE-cad-Fc is used in combination with the hN-cad-Fc.

[0015] In the specific embodiments of the various aspects of the present invention described above, the hE-cad-Fc and the hN-cad-Fc are immobilized on a matrix, preferably on the same matrix.

[0016] One aspect of the present invention relates to the use of hE-cad-Fc and hN-cad-Fc and their modified matrices for repairing cartilage layer and subchondral bone at cartilage defects after mediating stem cells to aggregate into stable multicellular aggregates.

[0017] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc and their modified matrices promote the phosphorylation of YAP protein and inhibit the phosphorylated YAP protein from entering the cell nucleus.

[0018] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc and their modified substrates inhibit β-catenin from entering the nucleus.

[0019] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc and their modified matrices can effectively inhibit the calcification of mature chondrocytes.

[0020] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc and their modified matrices promote the repair of cartilage and subchondral bone at cartilage damage by multicellular aggregates of stem cells.

[0021] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc fusion proteins promote the differentiation of stem cell multicellular aggregates into chondrocytes at the joint defect site and fill the damaged site in situ.

[0022] In one embodiment of the present invention, the hE-cad-Fc and hN-cad-Fc fusion proteins promote the secretion of anti-inflammatory factors by multicellular aggregates of stem cells under conditions of joint injury and inflammation.

[0023] Specifically, in the present invention, E-cadherin-Fc fusion protein and neural cell cadherin-Fc enhance the paracrine function of mesenchymal stem cells by upregulating the expression of endogenous cadherin, thereby significantly improving the therapeutic effect on colitis. Mesenchymal stem cells promote the polarization of macrophages from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype by producing immunosuppressive molecules such as prostaglandin E2, tumor necrosis factor-stimulating protein 6, and stanniocalcin, which is considered to be the basis for MSCs to treat inflammatory diseases. Mesenchymal stem cells have a direct effect on T cells by secreting indoleamine 2,3-dioxygenase, transforming growth factor, leukemia inhibitory factor, hepatocyte growth factor, interleukin 6, etc., which plays an important role in preventing autoimmune cells and inflammation from damaging normal tissues and cells and maintaining immune homeostasis.

[0024] E-cadherin-Fc fusion protein and neural cell cadherin-Fc fusion protein regulate the expression of endogenous cadherin in mesenchymal stem cells through spatiotemporal regulation, accelerate the initial mesenchymal condensation process, and enhance their ability to differentiate into chondrocytes. At the same time, by regulating the localization of β-catenin and YAP protein in cells, inhibiting the expression of osteoblast-related genes, and then inhibiting the calcification of differentiated mature chondrocytes, the differentiated mature chondrocytes can be filled in situ at the cartilage defect, promoting the repair of cartilage defect sites; and by promoting the paracrine function of mesenchymal stem cells, secreting immune regulatory factors, the role of restoring the homeostasis of the early inflammatory environment at the cartilage defect is achieved, accelerating the repair of the cartilage defect model.

[0025] In summary, the present invention provides the following embodiments:

[0026] 1. A modified matrix comprising E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein, and the matrix is ​​a hydrophobic microsphere.

[0027] 2. The modified matrix according to item 1, wherein the E-cadherin is human E-cadherin, and the Fc is the Fc of IgG (preferably IgG1); the N-cadherin is human N-cadherin, and the Fc is the Fc of IgG (preferably IgG1);

[0028] Preferably, the sequence of the E-cadherin is shown by SEQ ID NO: 2, and the sequence of the N-cadherin is shown by SEQ ID NO: 5.

[0029] More preferably, the Fc of the E-cadherin-Fc fusion protein and the N-cadherin-Fc fusion protein are the same or different, preferably the same, and more preferably the sequence shown in SEQ ID NO: 3.

[0030] More preferably, the E-cadherin-Fc fusion protein is preferably the sequence shown in SEQ ID NO:1; the N-cadherin-Fc fusion protein is preferably the sequence shown in SEQ ID NO:4.

[0031] 3. The modified matrix according to item 1, wherein the matrix is ​​PLGA microspheres, preferably PLGA / chitosan-heparin core-shell composite microspheres.

[0032] 4. A method for preparing a modified matrix, comprising modifying the matrix by mixing E-cadherin-Fc fusion protein and the N-cadherin-Fc fusion protein with the matrix.

[0033] 5. The preparation method according to item 4, wherein the matrix is ​​hydrophobic microspheres, preferably PLGA microspheres, more preferably PLGA composite microspheres modified with chitosan and heparin.

[0034] 6. A cell aggregate, which is an aggregate formed by the modified matrix according to any one of items 1 to 3 and cells.

[0035] 7. The cell aggregate according to item 6, wherein the cells are mesenchymal stem cells, iPS cells or embryonic stem cells in the blastocyst stage within 14 days, preferably mesenchymal stem cells, preferably derived from mammals, more preferably derived from humans, pigs or mice.

[0036] 8. The cell aggregate according to any one of items 6-7, wherein the ratio of the cells to the modified matrix is ​​3:1 to 10:1, preferably 3:1 or 5:1.

[0037] 9. Use of E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein or the modified matrix described in any one of items 1-3 or the cell aggregates described in items 6-8 in promoting cellular immune regulation function and / or promoting cartilage repair. Preferably, the E-cadherin-Fc fusion protein and the N-cadherin-Fc fusion protein are fixed on the same matrix or on different matrices. Preferably, the matrix is ​​hydrophobic microspheres (preferably PLGA microspheres, more preferably PLGA composite microspheres modified with chitosan and heparin).

[0038] 10. Use of E-cadherin-Fc fusion protein and N-cadherin-Fc fusion protein or the modified matrix described in any one of items 1 to 3 or the cell aggregate described in items 6 to 8 in the preparation of a medicament for treating the following diseases: colitis, arthritis, acute and chronic inflammation of soft tissue, and acute and chronic articular cartilage defects.

[0039] 11. The use described in item 9, wherein, when used to promote cellular immune regulatory function, the ratio of the E-cadherin-Fc fusion protein to the N-cadherin-Fc fusion protein is 1:5 to 5:1, preferably 1:3.

[0040] 12. The application described in Item 11, wherein the promotion of immune regulation includes enhancing the ability of paracrine-related factors (such as VEGF, TGF-β, IL-6, EGF, HGF, FGF-2, etc.), enhancing the expression of genes of anti-inflammatory factors (such as IL-1, IL-10 and TGFβ, etc.), and enhancing the regulatory ability of macrophages.

[0041] 13. The use described in item 9, wherein, when used to promote cartilage repair, the ratio of the E-cadherin-Fc fusion protein to the N-cadherin-Fc fusion protein is 1:1 to 1:3, preferably 1:2.

[0042] 14. The use described in item 13, wherein the promotion of cartilage repair includes promoting chondrocyte differentiation, inhibiting mature chondrocyte calcification, repairing joint defects and subchondral bone, etc.

[0043] References

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[0046] 3. Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing, Nature Communications, 9 (2018).

[0047] 4.Nanosphere-microsphere assembly: methods for core-shell materials preparation, Chemistry of materials, 13 (2001) 2210-2216.

[0048] 5. Biomimetic design of affinity peptide ligands for human IgG based protein A-IgG complex, Biochemical Engineering Journal, 88 (2014 (1-11). 6. FYWHCLDE-based affinity chromatography of IgG: Effect of ligand density and purifications of human IgG and monoclonal antibody, Journal of ChromatographyA, 1355 (2014) 107-114.

[0049] 7.Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media,Journal of ChromatographyA,1218(2011)1691-1700.

[0050] 8.Purification of human immunoglobulin G via Fc-specific smallpeptide ligand affinity chromatography, Journal of Chromatography A, 1216 (2009) 910-918.

[0051] 9. Mesenchymal stem cells: mechanisms of inflammation, Annual review ofpathology: mechanisms of disease, 6 (2011) 457-478. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Construction of chitosan-heparin complex, in which Figure 1 A is the infrared image of chitosan-heparin complexes with different ratios; Figure 1 B. Remaining chitosan and heparin in the solution after reaction of chitosan-heparin complexes with different ratios; Figure 1 C is the zeta potential diagram of chitosan-heparin complex solutions with different ratios.

[0053] Figure 2 :Hydrophilic treatment of microfluidic system chips, including Figure 2 A is the change of contact angle of glass chip before and after hydrophilic treatment; Figure 2 B is a light microscopy image of droplets prepared after hydrophilic treatment.

[0054] Figure 3 :The microfluidic system controls the droplet size by changing the continuous phase flow rate. Figure 3 A is the SEM image of the effect of the continuous phase flow rate on the microsphere particle size when the fixed dispersed phase is 0.4 mL / h; Figure 3 B is a particle size statistics diagram showing the effect of the continuous phase flow rate on the microsphere particle size when the stationary dispersed phase is 0.4 mL / h.

[0055] Figure 4 :The microfluidic system controls the droplet size by changing the dispersed phase flow rate. Figure 4 A is a SEM image showing the effect of dispersed phase flow rate on microsphere particle size when the stationary continuous phase is 0.5 mL / h; 4B is a particle size statistics graph showing the effect of dispersed phase flow rate on microsphere particle size when the stationary continuous phase is 0.5 mL / h.

[0056] Figure 5 :Morphological characterization of P / Ch composite microspheres prepared by microfluidics, Figure 5 A is the surface morphology observation SEM image of P / Ch composite microspheres; Figure 5 B is the SEM image of the section of P / Ch composite microspheres.

[0057] Figure 6 : Experiment on detecting the internal structure of P / Ch composite microspheres by fluorescence labeling.

[0058] Figure 7 :Characterization of the composition of P / Ch composite microspheres prepared by microfluidic system, Figure 7 A infrared detection image; Figure 7 B Thermogravimetric test diagram.

[0059] Figure 8 :Degradation performance characterization of P / Ch composite microspheres prepared by microfluidic system, Figure 8 A. pH change diagram of physiological saline in which composite microspheres are immersed during degradation; Figure 8 B is the graph showing the mass loss of composite microspheres during degradation; Figure 8 C is the SEM image of the composite microspheres during the degradation process.

[0060] Fig. 9 :Study on the release of growth factors from P / Ch composite microspheres prepared by microfluidic system, Fig. 9 A is the release curve of FGF-2 by composite microspheres; Fig. 9 B is the release curve of VEGF by composite microspheres.

[0061] Fig.10 :Study on cytotoxicity and hemolytic activity of P / Ch composite microspheres prepared by microfluidic system Fig.10 A is the detection of cytotoxicity of composite microspheres and hMSCs after culture on TCPS plates for 1, 3, and 7 days; Fig.10 B is the hemolytic test of the degradation solution after 14 days of microsphere degradation.

[0062] Fig.11 :The morphology of cell aggregates prepared by cells and P / Ch composite microspheres, Fig.11 A is the light microscopy image; Fig.11 B is the hematoxylin and eosin staining picture.

[0063] Fig.12 : Fluorescence staining of cell aggregates prepared by cells and P / Ch composite microspheres.

[0064] Fig.13 :The activity of cell aggregates without microspheres (control group), cell aggregates containing PLGA microspheres (MSC / PLGA group), cell aggregates containing P / Ch 2:1 composite microspheres (MSC / P / Ch 2:1 group) and cell aggregates containing P / Ch 4:1 composite microspheres (MSC / P / Ch 4:1 group) were studied, among which Fig.13 A is dead and alive staining; Fig.13 B is CCK-8 experiment; Fig.13 C is the gene expression of apoptosis markers Bax, Bcl-2 and β-actin in aggregates (PCR detection).

[0065] Fig.14 : Expression of stemness-related genes Sox-2, Nanog and β-actin genes in cell aggregates without microspheres (control group), cell aggregates with PLGA microspheres (MSC / PLGA group), cell aggregates with P / Ch 2:1 composite microspheres (MSC / P / Ch 2:1 group) and cell aggregates with P / Ch 4:1 composite microspheres (MSC / P / Ch 4:1 group) (PCR detection).

[0066] Fig.15 : Detection of gene levels of paracrine-related factors in cell aggregates without microspheres, cell aggregates containing PLGA microspheres, cell aggregates containing P / Ch 2:1 composite microspheres and cell aggregates containing P / Ch 4:1 composite microspheres (PCR detection).

[0067] Fig.16: Schematic diagram of the construction and identification of the pcDNA3.1 expression vector of hE-cad-Fc. Schematic diagram of the construction and identification of hE-cad-Fc. Fig.16 A shows the process of cloning cDNA encoding hE-cad and Fc domains in hE-cad-Fc into an expression vector. Fig.16 B represents gene restriction enzyme digestion identification of hE-cad-Fc fusion protein expression vector, M: protein standard; 1: restriction enzyme digestion of hE-cad-Fc fusion protein gene fragment; 2: restriction enzyme digestion of hE-cad-Fc extracellular domain gene fragment; 3: restriction enzyme digestion of Fc gene fragment; 4: undigested hE-cad-Fc plasmid vector; Fig.16 C represents WB identification of hE-cad-Fc fusion protein, M: protein standard; P: human anti-human IgG1; 1: reduced hE-cad-Fc fusion protein; 2: non-reduced hE-cad-Fc fusion protein.

[0068] Fig.17 : Schematic diagram of the construction and identification of the pcDNA3.1 expression vector of hN-cad-Fc. It includes a schematic diagram of the structure of pcDNA3.1-hN-cad-Fc and SDS-PAGE and WB analysis of purified hN-cad-Fc, where M represents protein standard; P represents human IgG (positive control, Sigma catalog number No. M15154); R represents hN-cad-Fc fusion protein purified under reduced state; N represents hN-cad-Fc fusion protein purified under non-reduced state.

[0069] Fig.18 :The immobilization amount, stability and distribution of hE-cad-Fc / hN-cad-Fc on the surface of P / Ch 2:1 composite microspheres.

[0070] Fig.19 : Release curve of IL-1β factor from P / Ch 2:1-IL-1β composite microspheres

[0071] Fig. 20 :Evaluation of biocompatibility of collagen, hE-cad-Fc, hN-cad-Fc and hE-cad-Fc / hN-cad-Fc combined modified P / Ch 2:1-IL-1β composite microspheres, among which Fig. 20 A is CCK-8 assay to evaluate the effects of microspheres with different surface modifications on the proliferation of hMSCs; Fig. 20 B is the blood compatibility evaluation of microspheres with different surface modifications; Fig. 20 C: The platelet adhesion of microspheres with different surface modifications was evaluated using scanning electron microscopy.

[0072] Fig.21:The morphology and related gene expression of cell aggregates prepared by different ratios of hMSCs cells and P / Ch 2:1-IL-1β microspheres modified with fusion protein surface.

[0073] Fig. 22 : Gene expression in cell aggregates containing P / Ch 2:1-IL-1β composite microspheres modified with different ratios of hE-cad-Fc and hN-cad-Fc.

[0074] Fig.23 : Observation of the morphology of cell aggregates without microspheres, stem cell aggregates containing collagen-modified P / Ch 2:1-IL-1β composite microspheres, hE-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres (MEP group), hN-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres (MNP group), and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) co-modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group).

[0075] Fig.24 : Cell aggregates without microspheres, stem cell aggregates containing collagen-modified P / Ch 2:1-IL-1β composite microspheres, hE-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres, hN-cad-Fc-modified P / C-h2:1-IL-1β composite microspheres, and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) co-modified P / C-h2:1-IL-1β composite microspheres, and the factor secretion function after 72 hours of culture.

[0076] Fig.25 : Cell aggregates without microspheres, stem cell aggregates containing collagen-modified P / Ch 2:1-IL-1β composite microspheres, hE-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres, hN-cad-Fc-modified P / C-h2:1 composite microspheres, and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) co-modified P / C-h2:1-IL-1β composite microspheres, after culturing for 72 hours, the expression of genes related to cellular immune regulation (PCR detection).

[0077] Fig.26: Cell aggregates without microspheres, stem cell aggregates containing collagen-modified P / Ch 2:1-IL-1β composite microspheres, hE-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres, hN-cad-Fc-modified P / C-h2:1-IL-1β composite microspheres, and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) co-modified P / C-h2:1-IL-1β composite microspheres, after culturing for 72 hours, the expression of endogenous cadherin in cells was detected. Fig.26 A is the endogenous cadherin gene expression in aggregate cells (PCR detection); Fig.26 B shows the expression of endogenous cadherin protein in the aggregates (WB detection).

[0078] Fig. 27 : Cell aggregates without microspheres, stem cell aggregates containing collagen-modified P / Ch 2:1-IL-1β composite microspheres (collagen group), hE-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres (MEP group), hN-cad-Fc-modified P / Ch 2:1-IL-1β composite microspheres (MNP group), and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) co-modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group), after culturing for 72 hours, the effect of aggregate cells on macrophage polarization, among which Fig. 27 A is a schematic diagram of co-culture; Fig. 27 B is TNF-α secreted by macrophages; Fig. 27 C, D show the expression of specific proteins of M1 and M2 macrophages; Fig. 27 E, F show the expression of genes related to the regulation of macrophage polarization by aggregate cells (PCR detection).

[0079] Fig.28 PBS injection (blank group), cell aggregates without microspheres (M group), and stem cell aggregates containing hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) and modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group) were implanted into the peritoneal cavity for the treatment of colitis mouse model. Fig.28 A shows the weight changes of mice with colitis; Fig.28 B is the disease activity index of colitis mice; Fig.28 C shows the colon morphology of mice with colitis; Fig.28 D is the colon length statistics of colitis mice.

[0080] Fig.29: Evaluation of the degree of colon lesions in mice with colitis treated with PBS injection (blank group), cell aggregates without microspheres (M group), and stem cell aggregates containing hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) combined with modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group). Fig.29 A is HE staining of colon sections of colitis mice; Fig.29 B is the pathological score of colon sections.

[0081] Fig.30 : Characterization of the immunomodulatory function of PBS injection (blank group), cell aggregates without microspheres (M group), and stem cell aggregates containing hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:3) and modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group) on the colitis mouse model. Fig.30 A is the CD4 in spleen of colitis mice + and CD8 + T cell flow cytometry scatter plot; Fig.30 B is the spleen CD8 of colitis mice + T cell statistics chart; Fig.30 C is the CD4 in spleen of colitis mice + T cell statistics chart; Fig.30 D is the CD4 + Scatter plot of T cell Th1 differentiation flow cytometry; Fig.30 E is the CD4 + Scatter plot of T cell Th17 differentiation flow cytometry; Fig.30 F is the CD4 + Statistical diagram of T cell Th1 differentiation; Fig.30 G is CD4 in the spleen of colitis mice + Statistical chart of T cell Th17 differentiation.

[0082] Fig.31 :Release curve of TGFβ1 factor from P / Ch 2:1-TGFβ1 composite microspheres.

[0083] Fig.32 : Cell morphology of hMSCs after chondrogenic differentiation induction on the surface of collagen, hE-cad-Fc / hN-cad-Fc concentration ratios of 1:1, 1:2 and 1:3, and hN-cad-Fc-modified polystyrene (PS) culture plates.

[0084] Fig.33:Effects of different ratios of hE-cad-Fc / hN-cad-Fc combined with matrix on chondrogenic differentiation of hMSCs. Cell aggregates prepared with collagen (MCP group), hE-cad-Fc / hN-cad-Fc concentrations of 1:1, 1:2, 1:3, and hN-cad-Fc (MNP group) modified P / Ch 2:1-TGFβ1 composite microspheres were induced to differentiate into chondrogenic tissues for 1 week. The expression of cartilage markers was significantly decreased after 1 week of induced differentiation. Fig.33 A is the gene expression of cartilage markers (PCR detection); Fig.33 B is the protein expression of cartilage markers (WB detection).

[0085] Fig.34 : Expression of N-cadherin in cell aggregates over time. Expression of endogenous N-cadherin in chondrogenic differentiation culture of cell aggregates without microspheres (M group), with collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres, among which Fig.34 A is the gene expression of endogenous N-cadherin (PCR detection); Fig.34 B is the protein expression of endogenous N-cadherin (WB detection).

[0086] Fig.35 : Differentiation efficiency of cell aggregates into chondrogenic differentiation. Expression of related genes and proteins in cell aggregates without microspheres (M group), containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 1, 2, 3 and 4 weeks of differentiation culture.

[0087] Fig.36 : Efficiency of cell aggregates differentiation into chondrogenic cells. Alcian blue staining results of cell aggregates without microspheres (M group), containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 1 and 2 weeks of differentiation culture.

[0088] Fig.37: The results of safranin fast green staining of cell aggregates without microspheres (M group), cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 1 and 2 weeks of differentiation culture.

[0089] Fig.38 : Expression and statistics of osteogenic-related genes in cell aggregates without microspheres (M group), cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 2 weeks of directed differentiation culture.

[0090] Fig.39 : Expression of endogenous E-cadherin and β-catenin in cell aggregates without microspheres (M group), cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 4 weeks of differentiation and culture. Fig.39 A, immunostaining of E-cadherin and β-catenin; Fig.39 Expression of β-catenin in the nucleus and cytoplasm of B cells and its statistical results.

[0091] Fig.40 : Evaluation of E-cadherin and YAP in cell aggregates without microspheres (M group), cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres after 4 weeks of differentiation culture; Fig.40 A, immunostaining of E-cadherin and YAP; Fig.40 B shows the protein expression and statistics of YAP and phosphorylated YAP in the nucleus and cytoplasm. hE-cad-Fc enhances the phosphorylation of YAP by promoting the expression of cellular E-cadherin, thereby inhibiting the entry of YAP into the nucleus.

[0092] Fig.41: After articular cartilage defect in rats, PBS was injected into the joint cavity. Cell aggregates without microspheres (M group) and cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres were injected into the joint cavity. The results of arthroscopic observation were performed 3 and 6 weeks after implantation.

[0093] Fig.42 : After articular cartilage defect in rats was repaired by intra-articular injection of PBS, cell aggregates without microspheres (M group) and cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres, and the results of safranin fast green staining of femoral tissue sections after 3 and 6 weeks of repair.

[0094] Fig.43 : After articular cartilage defect in rats, PBS was injected into the articular cavity, and cell aggregates without microspheres (M group) and cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres were repaired for 3 and 6 weeks. The repair effect of subchondral bone was detected by MicroCT. 43Aa is 3D imaging at 3 weeks, 43Ab is a cross-section of the injury site at 3 weeks, and 43Ac is a statistical graph of bone density and trabecular width at 3 weeks; 43Ba is 3D imaging at 6 weeks, 43Bb is a cross-section of the injury site at 6 weeks, and 43Bc is a statistical graph of bone density and trabecular width at 6 weeks.

[0095] Fig.44 : After articular cartilage defects in rats were intra-articularly injected with PBS, cell aggregates without microspheres (M group) and cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres. Immunostaining of human protein Ku80 and human and mouse type 2 collagen after 3 and 6 weeks of repair.

[0096] Fig.45: By adding IL-1β to the chondrogenic differentiation medium to simulate the arthritis microenvironment in vitro, the gene expression of anti-inflammatory factors IL-1, IL-10 and TGFβ in cell aggregates without microspheres (M group) and cell aggregates containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc / hN-cad-Fc (concentration ratio of 1:2) (MENP group) co-modified P / Ch 2:1-TGFβ1 composite microspheres were investigated. DETAILED DESCRIPTION

[0097] The present invention is described in more detail below by way of examples. However, it will be appreciated by those skilled in the art that the following examples are only for the purpose of illustrating the present invention and are not intended to limit the present invention.

[0098] The conventional chemical reagents used in the examples were all purchased from Solebow Biotech Co., Ltd.; the chips involved were produced by Dalian Weituo Co., Ltd., and the peptides involved were synthesized by Nanjing GenScript Biotech Co., Ltd.

[0099] Example 1. Synthesis of chitosan and heparin complex

[0100] The chitosan and heparin complex was prepared by ionic interaction as follows.

[0101] Accurately weigh 5 mg of heparin (from porcine intestinal mucosa, 185 units / mg, Aladdin) and add 1 mL of ddH 2 O, stir to fully dissolve. Accurately weigh 5 mg chitosan (deacetylation degree ≥ 95%, viscosity 100-200 mPa.s, Aladdin) and add 1 mL 1% acetic acid solution to fully dissolve; and use it to titrate 5 mg / mL heparin solution, react according to chitosan: heparin (volume) ratio of 1:1, 2:1, 3:1, 4:1, 5:1, incubate at room temperature for 30 min, centrifuge at 2000 × g for 5 min, discard the supernatant, and wash with ddH 2 O was washed twice, and the resulting white precipitate was chitosan-heparin complex; it was freeze-dried and used for infrared spectroscopy detection. Figure 1 The infrared spectrum of chitosan-heparin complex shown in A shows the IR spectrum of chitosan-heparin complex, where 1560 and 2818 cm -1 The characteristic peaks of stretching vibration of chitosan-NH are 1236, 1027 and 820 cm -1 It is the characteristic peak of S=O and COS stretching vibration of heparin, indicating that chitosan-heparin complex is formed by the combination of anion and cation.

[0102] Use dimethyl blue (DMB) and Orange Red II to detect the content of unreacted heparin and chitosan, respectively. Take 50μL of chitosan heparin reaction supernatant with different proportions and add 100μL to 10.7μg of DMB working solution, mix thoroughly, and measure the absorbance at 520nm. Use heparin solution of known concentration as standard curve to calculate the content of heparin in the supernatant. Take 50μL of chitosan heparin reaction supernatant with different proportions and add it to 20μL Orange Red II (1mM), mix thoroughly, centrifuge at 12000×g for 5min, take the supernatant and measure the absorbance at 484nm. Use chitosan solution of known concentration as standard curve to calculate the content of chitosan in the supernatant. Figure 1 B is the amount of unreacted chitosan and heparin in the reaction supernatant, that is, with the addition of chitosan, the content of unreacted heparin in the solution becomes less and less. When the ratio of chitosan to heparin is 3:1, the heparin content is the lowest, indicating that the chitosan added at this time completely titrates the heparin.

[0103] Take 100 μL, 200 μL, 300 μL, 400 μL and 500 μL of 5 mg / mL chitosan solution and add them to 100 μL of 5 mg / mL heparin solution to react fully, then add distilled water to a total volume of 750 μL, mix well and then perform Zeta potential detection. Figure 1 As shown in Figure C, with the increase of the chitosan-heparin reaction ratio, the potential of the complex changes from the initial negative potential when the heparin content is higher to the positive potential when the chitosan content is higher, and the potential tends to be stable at 3:1. Based on the above results, we chose the complex with a chitosan and heparin reaction ratio of 3:1 to prepare composite microspheres.

[0104] Example 2. Hydrophilic treatment of microfluidic chip

[0105] First, the chip microchannels were treated with 1M HCl and 1M NaOH solutions filtered through a 0.22 μm polyethersulfone filter for 30 min, and then rinsed with distilled water for 30 min to make the microchannels hydrophilic. After drying naturally, the contact angle was measured. Figure 2 The contact angle of the chip surface before hydrophilic treatment was 82.2°, and the contact angle of the chip surface after hydrophilic treatment was 13.4°, and the hydrophilicity of the chip was significantly improved. Figure 2 B. After hydrophilic treatment, droplets with good dispersion and uniform size can be generated in the microfluidic system, which are arranged in a matrix structure under an optical microscope. That is, hydrophilic treatment is conducive to the stable generation of uniform monodisperse droplets in the microfluidic system.

[0106] Example 3 Preparation of P / Ch composite microspheres using microfluidic technology

[0107] The preparation process of composite microspheres was optimized by using PLGA / chitosan-heparin (P / Ch) emulsion as dispersed phase and 2% polyvinyl alcohol solution as continuous phase:

[0108] 3.1 Preparation of two-phase solution in microfluidic system

[0109] Weigh 40 mg PLGA (L:G=50:50, Mw=25000, Jinan Daigang Bioengineering Co., Ltd.), dissolve in 1 mL dichloromethane (DCM), stir magnetically to fully dissolve, filter through a polyethersulfone microporous filter membrane for use. Resuspend the obtained chitosan-heparin complex in a sterile PBS solution, and treat it with high-speed ultrasound for 2 minutes to obtain a uniform suspension for use. Add 0.1 mL of the above chitosan-heparin complex suspension (containing 0 mg, 10 mg, and 20 mg of the complex, respectively) to 1 mL of the above PLGA / DCM solution, quickly place it on ice for high-speed ultrasound emulsification for 2 minutes, and use it as the dispersed phase of the microfluidic system. Weigh 2 g of polyvinyl alcohol (alcoholysis degree: 87.0-89.0%, viscosity: 4.6-5.4, Aladdin) and dissolve it in 100 mL of distilled water to prepare a 2% (w / v) PVA solution. After complete dissolution, filter it with a polyethersulfone filter membrane as the continuous phase of the microfluidic system for use (prepared and used immediately).

[0110] 3.2 Preparation of P / Ch composite microspheres using microfluidic system

[0111] The dispersed phase and the continuous phase were added to a 20 mL syringe (to remove bubbles), and the two phases were transported to the cross-shaped microfluidic chip using a syringe pump. After the droplets generated by the system were stable, the droplets were collected with a 2% PVA solution. At the same time, the DCM was volatilized by magnetic stirring, and the droplets were solidified to obtain composite microspheres. The composite microspheres were repeatedly washed with distilled water to remove PVA, and freeze-dried to obtain composite microspheres for SEM detection. Droplets of different particle sizes can be prepared by adjusting the flow rate of the two phases. Figure 4 As shown in Figure 2, when the continuous phase flow rate is fixed at 0.5 mL / h and the dispersed phase flow rate is changed, the microsphere particle size increases with the increase of the dispersed phase flow rate; at the same time, Figure 3 As shown in the figure, when the dispersed phase flow rate is fixed at 0.4 mL / h and the continuous phase flow rate is changed, within a certain range, the particle size of the obtained microspheres decreases with the increase of the continuous phase flow rate. When the flow rates of the continuous phase and the dispersed phase are maintained at 1.1 and 0.4 mL / h respectively, the particle size of the obtained composite microspheres is 23.5

[0112] μm. By the same method, the dispersed phase solution was changed to obtain composite microspheres of PLGA / chitosan-heparin complexes with different ratios (P / Ch 2:1, P / Ch 4:1, i.e., the ratios of PLGA and chitosan-heparin complexes were 40 mg:20 mg and 40 mg:10 mg, respectively); chitosan and heparin compounds were replaced with an equal volume of PBS solution, and hollow PLGA microspheres were prepared by the same method; growth factors were first combined with heparin, and then heparin / chitosan complexes were prepared, and P / Ch composite microspheres (i.e., microspheres loaded with growth factors) that released growth factors slowly were prepared by the same method.

[0113] Example 4. Characterization of the physical and chemical properties of P / Ch composite microspheres

[0114] The morphology, composition, internal structure, degradability and growth factor release characteristics of P / Ch composite microspheres with different compositions were systematically evaluated.

[0115] Resuspend the freeze-dried microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) in distilled water, blow evenly and then drop them onto the surface of a clean silicon wafer. Let them dry naturally and fix them on the stage with conductive glue and spray gold for 30 seconds. Observe them under SEM. Figure 5 As shown in A: P / Ch composite microspheres are uniform in size and have a smooth surface.

[0116] The freeze-dried microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) were resuspended in OCT, completely frozen at -20℃, and sliced ​​with a cryostat to a thickness of 8μm and attached to a cover glass. After washing off the OCT with distilled water, the microspheres were naturally dried and fixed with conductive glue to the stage for gold spraying and SEM observation. Figure 5 As shown in B: The P / Ch composite microspheres present a core-shell structure with a uniform shell thickness of about 1-2 μm.

[0117] Preparation of FITC-labeled chitosan, namely weigh 2 mg of chitosan and dissolve it in 1% acetic acid solution, add 10 μL of 1 mg / mL FITC solution while stirring, protect from light throughout the process, and react on ice at 4°C for 24 hours; put the reaction solution into a dialysis bag (molecular weight is 8000), dialyze in distilled water (change the dialysate every two days), after two weeks of dialysis, take out and freeze-dry it, and store it at 4°C for later use. The oil-soluble dye Nile red is used for PLGA labeling, that is, Nile red is directly dissolved in the DCM solution of PLGA (1 mg / mL), and the chitosan / heparin complex is prepared by using the FITC-labeled chitosan and heparin, and the fluorescently labeled composite microspheres are prepared according to the method in Example 3. The prepared fluorescently labeled composite microspheres are added dropwise to a special confocal dish and observed under a laser confocal microscope. Figure 6As shown: the FITC-labeled chitosan / heparin complex is located inside the microspheres, and the Nile red-labeled PLGA is located outside the microspheres. As the amount of chitosan / heparin complex increases, its distribution in the microspheres increases.

[0118] At room temperature, completely dried microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) were mixed with potassium bromide and completely ground, then pressed into tablets, and scanned and analyzed using an infrared spectrometer. Potassium bromide tablets were used as blank controls. Figure 7 A is the infrared spectrum of microspheres. The characteristic peaks of chitosan, heparin and PLGA can be clearly observed in the infrared spectra of P / Ch 4:1 and P / Ch 2:1 composite microspheres. -1 It is the characteristic peak of heparin S=O, CO, COS, 1560cm -1 is the characteristic peak of chitosan-NH, 1755cm -1 is the stretching vibration peak of PLGA.

[0119] At room temperature, weigh an equal amount of completely dried microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) and add them to the sample tank of the thermogravimetric analyzer for detection. Figure 7 As shown in B: All samples showed a typical two-stage or two-step decomposition trend, corresponding to two endothermic peaks. A small mass loss before 100°C was the decomposition of the adsorbed water in the sample. In the second step of degradation, a relatively large mass loss occurred, which was due to the decomposition of the polymer itself. Pure PLGA decomposed at around 280°C and decomposed completely at 370°C, with almost 100% weight loss. Chitosan began to thermally decompose at 220°C, losing about 50% weight at 370°C, and pure heparin thermal decomposition began at around 215°C. The results showed that with the increase of the amount of chitosan / heparin in the components, the mass loss at 450°C decreased in turn, with P / Ch 4:1 reducing the most, followed by P / Ch 2:1, that is, during the thermal decomposition process, only PLGA was completely decomposed, leaving more chitosan and heparin.

[0120] Weigh equal amounts of microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) and place them in 10 mL centrifuge tubes, add equal amounts of physiological saline, and incubate in a 37°C constant temperature shaker. Samples were taken every three days to test the pH of the extract. The microspheres were freeze-dried and weighed, and the morphology was observed using SEM. The mass loss was calculated using the following equation: Mass loss (%) = (M 0 -M d ) / M 0 × 100%. 0 is the initial mass of the microsphere, M d is the mass of the microspheres after degradation. Figure 8A is the pH change curve of the saline solution in which the microspheres were immersed. It can be seen that the pH value of the pure PLGA extract decreased the fastest, that is, the lactic acid and glycolic acid produced by the degradation of PLGA caused its pH value to decrease rapidly, while the composite microspheres decreased more slowly, indicating that the presence of chitosan alleviated the acidic environment produced by the degradation of PLGA. On the 15th day of the observation period, the pH value of the PLGA extract decreased to 6.23, while the extract of the composite microspheres remained neutral, especially when the ratio of PLGA to chitosan heparin complex was P / Ch 2:1, the pH value of its extract was 7.20, that is, the amino groups in the chitosan structure alleviated the acidity produced by the degradation of PLGA to a certain extent.

[0121] Figure 8 B is the mass loss diagram of microsphere degradation. It can be seen that the mass loss of pure PLGA microspheres is greater than that of P / Ch 4:1 and P / Ch 2:1 composite microspheres in the same time. Figure 8 C is the SEM images of degradation at different time points. As shown in the figure, the surface of PLGA microspheres began to become rough on the 3rd day of degradation, obvious gaps appeared on the surface on the 6th day, and they collapsed and lost their spherical structure on the 9th day. The spherical structure no longer existed on the 12th day. The degradation of P / Ch composite microspheres was slower than that of PLGA microspheres. It can be seen that the microsphere structure can still be observed on the 12th day. We believe that the improvement of the acidic microenvironment in the composite microspheres partially slowed down the acid-accelerated disintegration rate during the degradation of PLGA.

[0122] 2 mg of growth factor-loaded microspheres (PLGA, P / Ch 4:1, P / Ch 2:1) were suspended in 0.5 mL PBS (0.02% Tween 20 and 10 mg / mL BSA) and incubated in a 37°C constant temperature incubator, and the concentration of growth factors (FGF-2, VEGF) in the extract was continuously detected. 10 μL of supernatant was collected by centrifugation every 3 days, and 10 μL PBS (0.02% Tween 20 and 10 mg / mL BSA) was added to continue incubation. The supernatant was stored at -20°C. After collection, the enzyme-linked immunosorbent assay kit (ELISA) was used to measure the content of growth factors. The concentration of growth factors was obtained by comparison with the standard curve, and all experiments were repeated three times. Fig. 9 As shown in the figure: the growth factors loaded by PLGA microspheres showed a significant burst release (40%) at the beginning, and were basically released completely on the 9th day. However, the P / Ch 4:1 and P / Ch 2:1 composite microsphere groups showed a sustained release trend, with factors continuously released within 15 days.

[0123] Example 5. Two-dimensional co-culture of P / Ch composite microspheres and cells

[0124] The composite microspheres were co-cultured with umbilical cord mesenchymal stem cells (hMSCs) in two dimensions to study the cytotoxicity and hemolytic activity of the composite microspheres under two-dimensional conditions.

[0125] hMSCs (Guangzhou Saiye Biotechnology Co., Ltd.) in good growth condition were treated with trypsin at 37°C for 1 min, and digestion was terminated with DMEM / F12 medium containing 10% fetal bovine serum (FBS, BI, USA). The cells were centrifuged at 1000×g for 5 min, the supernatant was discarded, and 200μL DMEM / F12 medium was added to prepare a high-density cell suspension.

[0126] hMSCs were mixed with PLGA, P / Ch 4:1, and P / Ch 2:1 composite microspheres at a ratio of 1:1, and added to TCPS culture plates for co-culture. CCK-8 working solution (10:1) was added on days 1, 3, and 5, and incubated at 37°C for 4 h. After sufficient shaking and mixing, 100 μL was taken to measure the absorbance at 450 nm. hMSCs cultured in two dimensions under the same conditions were used as the control group. The results are shown in Figure 2. Fig.10 As shown in A, with the extension of culture time, the P / Ch 2:1 group showed better effect in promoting cell proliferation. Fresh blood of 4-week-old ICR mice was collected in an EDTA-treated centrifuge tube and centrifuged and washed thoroughly with sterile saline until the supernatant was transparent and clear. The red blood cells were diluted to 1×10 9 800 μL of the 15-day degradation solution of PLGA, P / Ch 4:1, and P / Ch 2:1 composite microspheres were mixed with 200 μL of red blood cell suspension, placed in a 37°C constant temperature incubator for 4 hours, and the same amount of ultrapure water was used as the positive control and saline as the negative control. Then centrifuge at 10,000 × g for 5 minutes, gently remove the centrifuge tube, observe and take pictures. Take 100 μL of the supernatant in a clean 96-well plate and detect the OD value at 577 nm under an enzyme marker. Calculate the hemolysis rate (%) according to the following formula = [(OD sample-OD negative) / (OD positive-OD negative)] × 100. Fig.10 B is the result of the hemolysis test. It can be seen that when pure water is used as the positive control, the PLGA group has a slight hemolysis phenomenon, while the supernatant of the P / Ch 4:1 and P / Ch 2:1 groups is colorless and transparent, and no hemolysis occurs. The above results show that the acidic microenvironment produced by PLGA degradation in the P / Ch 2:1 composite microspheres is alleviated, the blood and cell compatibility of the composite microspheres is improved, and cell proliferation is promoted.

[0127] Example 6. Preparation of P / Ch composite microspheres and cell aggregates

[0128] Utilize low adhesion Aggrewell TMThe principle of spontaneous aggregation of hMSCs into aggregates in culture plates TM Anti-Adherence Rinsing solution was added to the culture plate, centrifuged at 2000×g for 5 min, the solution was discarded, and the plate was rinsed with PBS for later use. The sixth generation hMSCs in good condition were digested from the culture flask and 8×10 5 The cells and microspheres (PLGA, P / C-h 4:1, P / Ch 2:1) were fully mixed in a ratio of 3:1, and the cell and microsphere suspension (600 cells / microwell) was added to the treated Aggrewell TM The plate was centrifuged at 1000×g for 5 min at room temperature, and then placed in a 37°C incubator for 12 h. The formation of cell aggregates was observed under a microscope. hMSCs aggregates with added PLGA microspheres (expressed as MSC / PLGA), hMSCs aggregates with added P / Ch 4:1 microspheres (expressed as MSC / P / Ch 4:1), hMSCs aggregates with added P / C-h2:1 composite microspheres (expressed as MSC / P / Ch 2:1), and hMSCs aggregates without added microspheres (expressed as MSC) were used as the control group. Fig.11 A shows the results of an inverted phase contrast microscope, showing aggrewell TM The MSC, MSC / PLGA, MSC / P / Ch 4:1, and MSC / P / Ch 2:1 groups in the culture plate can all form aggregates with clear edges and complete structures. The size of the aggregates between different groups is basically the same, indicating that the composition of the microspheres does not affect the formation and morphology of cell aggregates at the current optimized ratio.

[0129] The prepared aggregates were suspended in 1.5% (w / v%) sodium alginate solution and added dropwise to 0.1 M sterile CaCl 2 Solution, incubate at room temperature for 5 min until a stable gel is formed, and then absorb the CaCl 2 Solution, washed 3 times with physiological saline, and then added to 4% paraformaldehyde for fixation for more than 2 days. The fixed aggregates were placed in an automatic dehydrator for dehydration, embedded in a paraffin embedding machine, sliced ​​in a microtome (thickness 3.5μm), baked at 65℃ for 4h, dewaxed in xylene for 10min, rehydrated with gradient ethanol, stained with hematoxylin for 2min, rinsed with running water for 5min, differentiated with hydrochloric acid and alcohol for 2s, stained with eosin for 1min, rinsed with running water for 5min, dehydrated with gradient ethanol, transparentized with xylene for 10min, and after the xylene was completely dried, the slices were sealed with neutral gum and observed on the machine. The results are as follows Fig.11 As shown, the microspheres were encapsulated by cells and evenly distributed within the aggregates.

[0130] Composite microspheres were prepared according to the method in Example 3. Nile red (1 μg / mL) was added to DCM during the preparation to prepare red fluorescently labeled composite microspheres, which were freeze-dried for later use. Cell aggregates were prepared using red fluorescently labeled composite microspheres and hMSCs according to the method in Example 5. The prepared cell aggregates were immersed in 4% paraformaldehyde for fixation for 2 days, incubated with 1% TritonX-100 for 1 hour, and blocked with 1% BSA at room temperature for 1 hour. After blocking, FITC-labeled phalloidin diluted with 1% BSA was added and incubated at room temperature in the dark for 1 hour, and then DAPI staining solution was added and incubated at room temperature in the dark for 30 minutes. After the incubation, the cells were washed 3 times with PBS and observed under a laser confocal microscope. The results are shown in FIG. Fig.12 As shown, consistent with the results of paraffin sections, the microspheres can be evenly distributed inside the aggregates.

[0131] Example 7. Study on the effect of P / Ch composite microspheres on the activity of aggregated cells

[0132] Prepare cell aggregates according to the method of Example 6, suspend them in calcium alginate hydrogel, add DMEM / F12 solution containing 10% FBS, and change the solution every 3 days in a 37°C incubator. On the 1st, 3rd and 7th days of culture, discard the culture medium, add 0.1M sterile EDTA solution, shake slightly, and after the calcium alginate hydrogel is lysed, centrifuge at 1000×g for 5 minutes, discard the supernatant, wash 3 times with PBS, and recover the cell aggregates. First, use the live-death staining method to examine the survival of cells in the aggregates. Prepare FDA / PI cell live-death staining solution according to the instructions, dissolve FDA in acetone to prepare a 5mg / mL mother solution, and dissolve PI in PBS to prepare a 100μg / mL mother solution for standby use. Add 1:100 diluted FDA and 1:200 diluted PI working solution to the cell aggregates, incubate in a light-proof shaker at room temperature for 5 minutes, then let it stand. When the aggregates completely sink to the bottom of the centrifuge tube, remove the dye solution, wash twice with PBS, 5 minutes each time, then transfer to a culture dish and observe on the microscope. Fig.13 As shown in A, a small amount of apoptotic cells were observed in the MSC group aggregates and the MSC / PLGA group aggregates on the 3rd day of culture. On the 7th day, a large number of cells stained by PI were observed in the MSC group aggregates and the MSC / PLGA group aggregates, indicating that obvious apoptosis had occurred, while no obvious apoptosis was observed in the MSC / P / Ch 2:1 group aggregates.

[0133] The cell viability in the aggregates was detected using the CCK-8 method. The specific operation was to culture the recovered cell aggregates in a low-adhesion plate and measure the cell viability on the 1st, 3rd and 7th day according to the method in Example 5. The results are shown in FIG. Fig.13As shown in B. Consistent with the results of FDA / PI staining, the cells in the aggregates of MSC / P / Ch 4:1 and MSC / P / Ch 2:1 groups showed a trend of promoting cell proliferation, and the cell activity of MSC / PLGA aggregates decreased, indicating that the acidic environment of PLGA degradation is not conducive to maintaining the activity of cells in three-dimensional aggregates.

[0134] On days 1, 3, and 7 of culture, cell aggregates were obtained according to the method in Example 6. Every 500 aggregates (3×10 5 Cells) were added with 1 mL of Trizol to extract total RNA. The specific steps were as follows: 200 μL of chloroform solution was added to each 1 mL of Trizol used to treat cells, the mixture was mixed by inversion and allowed to stand at room temperature for 3 minutes, and centrifuged at 4°C 12000×g for 15 minutes. The colorless water layer was carefully aspirated into a new centrifuge tube with a pipette, and then 500 μL of isopropanol was added to each tube, the mixture was mixed by inversion and allowed to stand at room temperature for 10 minutes, and centrifuged at 4°C 12000×g for 15 minutes. The supernatant was discarded, 1 mL of 75% ethanol was added, vortexed briefly, and centrifuged at 7500×g for 5 minutes. The supernatant was discarded, dried at room temperature, and then precipitated with 20 μL of RNase-free ddH 2 Dissolve RNA in 55-60℃ water bath. After RNA is completely dissolved, check the concentration in BioDropμLIFE and immediately convert RNA into cDNA. The specific operation is as follows: Prepare the system in Table 1 in an RNase-free centrifuge tube:

[0135] Table 1

[0136]

[0137] After mixing the reagents in the table above, centrifuge quickly, set the temperature in the PCR instrument to 55℃ for 30min, 85℃ for 5min, and 4℃∞ for reverse transcription, and store the obtained cDNA in a -20℃ refrigerator for later use. Then perform semi-quantitative PCR detection and configure the reaction system according to Table 2:

[0138] Table 2

[0139]

[0140] After adding the components in the table in sequence, centrifuge quickly, and react in the PCR instrument according to the program to obtain the PCR product. Add DNA loading buffer to the reaction product and mix it thoroughly. Weigh 1g of agarose powder, dissolve it in 100mL TAE solution, and heat it in a microwave oven to obtain agarose gel. After the gel is slightly cool, add 10μL Gel Red and mix well, then pour it into the gel tank, wait for the gel to completely solidify, and then apply the sample. Adjust the voltage to 140V, after 30min of electrophoresis, take out the gel, image it, and the PCR primer sequence is shown in Table 3. The results are as follows Fig.13 As shown in Figure C, on the third day of culture, the expression of the pro-apoptotic gene Bax could be detected in the MSC group, and a small amount of Bax was also expressed in the MSC / PLGA group, while there was no expression of pro-apoptotic genes in the P / Ch 2:1 group, and its anti-apoptotic gene Bcl-2 was significantly expressed. This shows that cell apoptosis has occurred in the MSC group and the MSC / PLGA group at this time, and no obvious cell apoptosis has occurred in the MSC / P / Ch 2:1 group. On the seventh day of culture, the expression of pro-apoptotic genes in the MSC and MSC / PLGA groups was further enhanced, and the expression of pro-apoptotic genes was still not detected in the MSC / P / Ch 2:1 group, and the anti-apoptotic genes continued to be highly expressed.

[0141] Table 3

[0142]

[0143] Example 8 Study on the Effect of P / Ch Composite Microspheres on the Biological Function of Aggregate Cells

[0144] The effects of composite microspheres on the stemness and paracrine properties of cell aggregates were systematically characterized.

[0145] A series of cell aggregates were prepared according to the method of Example 6 and cultured normally for 7 days. RNA was extracted according to the method of Example 7 on the 1st, 3rd and 7th days of culture, and the expression of stemness-related genes (Sox-2, Nanog) was detected by PCR. The results are as follows: Fig.14 As shown in the figure, with the extension of in vitro culture time, the expression of stemness genes in the aggregates of MSC group and MSC / PLGA group gradually decreased. The stemness genes in the aggregates of MSC / P / C-h2:1 group continued to be highly expressed, indicating that MSC / P / Ch 2:1 composite microspheres can better maintain the stemness of hMSCs inside the aggregates.

[0146] Prepare cell aggregates according to the method of Example 6, and recover aggregates according to the method of Example 6 on days 1, 3, and 7 of culture. For the gene level of paracrine factors in the aggregates, extract total RNA according to the method of Example 7, reverse transcribe to obtain cDNA, use cDNA as a template, follow the instructions of Roche Realtine PCR kit, add the components in Table 4 in sequence, centrifuge quickly, place in fluorescent quantitative PCR instrument, and detect according to the program of 95℃ 5min×1; 95℃ 30s, annealing temperature 1min, 72℃ 1min×35; 72℃ 5min×1; 4℃∞. The data obtained are based on the internal reference β-actin of each group, according to 2 -△△Ct Data statistics were performed and the PCR primer sequences are shown in Table 5.

[0147] Table 4

[0148]

[0149] The results are as follows Fig.15 As shown, with the extension of culture time, the expression level of paracrine factors of cells in the MSC / P / Ch 2:1 group continued to increase, indicating that the composite microspheres can maintain the high level of expression of paracrine factors of cells inside the aggregates.

[0150] Table 5

[0151]

[0152] Example 9: Construction and expression of human hE-cad-Fc fusion protein

[0153] The construction method is described in Xu Jianbin's doctoral dissertation, Nankai University, December 2013 (Xu Jianbin, Research on the construction of stem cell microenvironment by human E-cadherin fusion protein matrix [D]. Tianjin. School of Life Sciences, Nankai University, 2013: 1-145.). The main steps are as follows.

[0154] 9.1 Cloning and sequence analysis of the E-cadherin extracellular domain gene

[0155] According to the human E-cadherin protein sequence and functional division included in the UniProt database, specific PCR primers were designed in combination with the gene sequence included in GenBank (NCBI Reference Sequence: NM 004360.3) to amplify the extracellular region of the E-cadherin protein. The primer sequences are shown in Table 6.

[0156] Table 6

[0157]

[0158] Total mRNA extraction from L-02 cells (purchased from the cell bank of the Chinese Academy of Sciences Typical Culture Collection Committee): mRNA was extracted according to the conventional method of the Molecular Cloning Experiment Guide (3rd edition). OD value was measured to quantify RNA purity and concentration. Reverse transcription was performed according to the method in Example 7. L-02 mRNA was used as a template for reverse transcription to form cDNA, and the E-cadherin gene fragment was amplified. The PCR reaction system is shown in Table 7:

[0159] Table 7

[0160]

[0161] The amplification conditions were as follows: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles, and a final extension at 72°C for 10 min. 380 μL ddH 2O, then extract once with an equal volume of phenol / chloroform / isoamyl alcohol, add 1 / 10 volume of 3M NaAc (pH 5.0) and 2 volumes of anhydrous ethanol, place at -20℃ for 1h; centrifuge at 12000g for 10min at 4℃, wash the DNA precipitate twice with 70% ethanol, dry in vacuum, and dissolve the precipitate in an appropriate amount of TE.

[0162] 9.2 Construction of pcDNA3.1-hE-cad-Fc eukaryotic expression vector

[0163] (1) PCR product purified by Hind III and Not I double digestion

[0164] The enzyme digestion system is as follows:

[0165] Table 8

[0166]

[0167] The reaction was carried out at 37°C overnight, the enzyme was inactivated at 65°C for 15 min, and 350 μL ddH 2 O, then extracted once with an equal volume of phenol / chloroform / isoamyl alcohol, added 1 / 10 volume of 3M NaAc (pH5.0), 2 volumes of anhydrous ethanol, and placed at -20°C for 1 hour. Centrifuged at 4°C, 12000×g for 10 minutes, the DNA precipitate was washed twice with 70% ethanol, vacuum dried, and the precipitate was dissolved in 10μ TE.

[0168] (2) Hind III and Not I digestion of pcDNA / 3.1;

[0169] Table 9

[0170]

[0171] The reaction was allowed to proceed overnight at 37°C. The digestion products were separated by electrophoresis in 1% agarose gel, and the target fragments were cut out under UV light and recovered using a DNA agarose gel recovery kit (TaKaRa). The recovered fragments were dissolved in 25 μL ddH 2 In O.

[0172] (3) Ligation reaction between vector and target fragment and transformation reaction

[0173] The reaction system is as follows:

[0174] Table 10

[0175]

[0176] 16℃ for 16h. Then CaCl 2Transform competent cells BL21(DE3) and culture overnight at 37℃ for 16-18h. Pick transformants and extract a small amount of plasmid for detection.

[0177] The recovered target gene E-cadherin extracellular region and the vector pcDNA3.1 with Fc fragment were double-digested (Hind III and Not I) at a constant temperature of 37°C. After electrophoresis gel recovery, the recovered products were mixed and ligated at 16°C overnight under the catalysis of T4 DNA ligase. After the ligation products were transformed into E.coli DH5α competent cells, ampicillin (Amp + ) for resistance screening. After extracting the plasmid, double enzyme digestion was performed for identification, and the correct recombinant plasmid was initially identified for DNA sequence analysis ( Fig.16 A). The constructed recombinant plasmid was named pcDNA3.1 / hE-cad-Fc. The sequence was verified to be correct by sequencing. The sequence of hE-cad-Fc fusion protein is shown in SEQ ID NO: 1, wherein the sequence of hE-cad is shown in SEQ ID NO: 2, and the sequence of Fc is shown in SEQ ID NO: 3.

[0178] 9.3 Cell transfection and protein purification

[0179] pcDNA3.1 / hE-cad-Fc was transfected into 293F cells (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences).

[0180] The specific binding of immunoglobulin Fc fragment to rProtein A was used to purify the target protein using Hitrap rProtein A FF column from GE Healthcare.

[0181] 9.4 Western blotting (WB) analysis

[0182] The purified hE-cad-Fc fusion protein was transferred to a PVDF membrane after electrophoresis on a 10% SDS-PAGE gel, blocked with 5% skim milk for 2 hours, incubated with a rabbit anti-human E-cadherin extracellular domain monoclonal antibody (RD, USA, 1:400 dilution) at 4°C overnight, and incubated with a HRP-labeled goat anti-rabbit secondary antibody (abcam, USA, 1:10000 dilution) at room temperature for 1 hour, washed with TBST, and exposed to DAB reagent for development and fixation. β-mercaptoethanol was not added to the loading buffer when detecting Fc dimer formation. The results are shown in Fig.16 As shown in B, a band can be seen at ∼240 KD in the non-reduced state and a band can be seen at ∼120 KD in the reduced state. These results suggest that the hE-cad-Fc fusion protein exists in the form of a dimer.

[0183] Example 10. Construction and expression of human hN-cad-Fc fusion protein

[0184] The sequence of the hN-cad-Fc fusion protein is shown in SEQ ID NO: 4, wherein the sequence of SEQ ID NO: 5 represents the sequence of hN-cad; and the sequence of SEQ ID NO: 3 represents the sequence of Fc.

[0185] Table 11

[0186]

[0187] According to the method in Example 9, human hN-cad-Fc fusion protein was constructed and expressed, and the cloning and sequence analysis of the extracellular region gene N-cadherin of neural cell cadherin were performed in sequence. According to the human N-cadherin protein sequence and functional partitioning included in the UniProt database, specific PCR primers were designed in combination with the gene sequence included in GenBank (NCBI Reference Sequence: NM_001795.3), and the hN-cadherin protein extracellular region (EC1-EC5) was amplified. The upstream primer introduced the EcoRⅤ restriction site (underlined), and the downstream primer introduced the NotⅠ restriction site (underlined). Total mRNA was extracted from neural cells (ScienCell), the pcDNA3.1-hN-cad-Fc eukaryotic expression vector was constructed, cell transfection and protein purification were performed, and WB analysis was performed. The results are shown in FIG. Fig.17 As shown, a band can be seen at ∼240 KD in the non-reduced state and a band can be seen at ∼120 KD in the reduced state. These results suggest that the hN-cad-Fc fusion protein is in the form of a dimer.

[0188] Example 11. Immobilization and stability detection of fusion protein on the surface of P / Ch 2:1 composite microspheres

[0189] The microspheres in this experimental example are all P / Ch 2:1 composite microspheres prepared according to the method in Example 3. Type I collagen (collagen, BD, USA, Cat. No. 354249) was diluted to a final concentration of 20 μg / mL with PBS buffer (pH=7.20), and hE-cad-Fc, hN-cad-Fc and hE-cad-Fc / hN-cad-Fc mixed solution (hE-cad-Fc to hN-cad-Fc ratio of 1:3) were diluted to a final concentration of 20 μg / mL, and 1 mg of P / Ch composite microsphere powder was soaked in the above mixed solution, and then placed in a 37°C horizontal shaker and incubated at 150×g for 2h, the supernatant was discarded, and the fusion protein that was not stably fixed was washed with PBS buffer (3 times). Then, the fixed amount was detected by adding 300 μL of 5% BSA solution, placing it in a 37°C horizontal shaker for blocking at 150×g for 2 h, and then adding 100 μL of diluted HRP-labeled goat anti-human IgG (Abcam, USA) at a dilution ratio of 1:10000, placing it in a 37°C horizontal shaker for blocking at 150×g in the dark for 1 h, washing with PBS buffer 5 times, adding 300 μL of TMB (Solebo, product number PR1200) colorimetric solution to each tube, placing it in a 37°C horizontal shaker for reaction at 150×g in the dark for 30 min, adding 300 μL of stop solution, taking 200 μL of the solution and adding it to a 96-well plate, and measuring the absorbance at 452 nm to determine the maximum fixed amount of the three fusion proteins co-immobilized on the surface of the microspheres. Subsequently, hE-cad-Fc and hN-cad-Fc were co-immobilized on the surface of the composite microspheres at the maximum immobilization amount P / Ch 2:1 according to the above method, and then immersed in PBS buffer, DF12 medium and DF12 + 10% FBS for 28 days, and then the absorbance of the supernatant was detected at 0, 3 and 5 days according to the above method to evaluate the fixation time of the fusion protein on the microsphere surface. TM 550Antibody Labeling Kit (ThermoFisher, Cat. No. 84530) and DyLight TM 488 Microscale Antibody Labeling Kit (ThermoFisher, Cat. No. 53025) was used to label hE-cad-Fc and hN-cad-Fc (labeling process is as follows), and then three fluorescently labeled proteins were fixed on the surface of P / Ch 2:1 composite microspheres according to the maximum fixation amount, and the distribution of fusion proteins on the surface of microspheres was observed using a laser confocal microscope. The results are shown in Fig.18As shown, the two fusion proteins can be fixed on the surface of the microspheres at the same time; when immersed in commonly used PBS buffer, culture medium or culture medium containing serum for a period of time, it can be found that the absorbance of the supernatant does not change much, indicating that the fusion protein can stably exist on the surface of the microspheres for up to 5 days; by fixing the fusion protein with fluorescent dye on the surface of the microspheres and using laser confocal detection, it can be found that the two fusion proteins can be evenly distributed on the surface of the microspheres.

[0190] The fusion protein fluorescence labeling step:

[0191] 1. Take 40 μL of 0.67 M borate buffer and mix evenly with 500 μL (2 mg / mL) fusion protein solution;

[0192] 2. Add the above solution to a test tube pre-filled with 50 μg of fluorescent dye, vortex gently and mix well with a pipette, and incubate at room temperature for 60 minutes away from light;

[0193] 3. Take 250 μL of the evenly mixed adsorption resin and place it in a centrifuge tube containing a centrifuge column. Centrifuge at 1000×g for 1 min, discard the supernatant, and transfer the centrifuge column to a new centrifuge tube.

[0194] 4. Add 250 μL of the solution after the reaction in step 2 above to the centrifugal column in step 3, and vortex lightly to mix the reaction solution with the adsorption resin;

[0195] 5. Centrifuge at 1000 g for 1 min, collect the purified solution to complete the labeling of the fusion protein, and store it at 4°C or -80°C in the dark.

[0196] Example 12. Detection of IL-1β release ability of fusion protein surface modified P / Ch 2:1-IL-1β composite microspheres

[0197] P / Ch 2:1 composite microspheres loaded with IL-1β (P / C-h2:1-IL-1β) were prepared according to the method in Example 4, and the IL-1β release performance of P / Ch 2:1-IL-1β microspheres was tested according to the method in Example 4. Fig.19 The IL-1β loaded on the P / Ch 2:1 composite microspheres modified with the fusion protein surface showed a sustained release trend, and the factor was controllably released within 15 days.

[0198] Example 13. Cytotoxicity detection of fusion protein surface modified P / Ch 2:1-IL-1β composite microspheres

[0199] First, hMSCs cells and P / Ch 2:1-IL-1β composite microspheres modified with fusion protein were mixed at a ratio of 1:1, and 1×10 4The cells were seeded into a 96-well plate at a density of 1.5 μg / mL. CCK-8 solution was added at 24, 48, and 72 h to detect cell activity and hemolysis according to the method in Example 5. hMSCs cultured in two dimensions under the same conditions were used as the control group. The results are shown in Table 5. Fig. 20 As shown in the figure, when hMSCs were co-cultured with microspheres modified with fusion protein surface, compared with the control group, the microspheres modified with fusion protein surface had no significant effect on the proliferation activity of cells, and the microspheres modified with fusion protein surface did not cause obvious hemolysis. In addition, it can be seen that the microspheres modified with fusion protein surface can inhibit the adhesion and activation of platelets. The above comprehensive explanation shows that the microspheres modified with fusion protein surface have good cell and blood compatibility.

[0200] Example 14. Determination of the mixing ratio of fusion protein surface modified P / Ch 2:1-IL-1β composite microspheres and cells

[0201] 8×10 5 hMSCs cells and fusion protein surface modified P / Ch 2:1-IL-1β composite microspheres were mixed in a ratio of (3:1, 5:1, 8:1, 10:1), and aggregates were prepared according to the method of Example 6. The cells were cultured in DMEM / F12 medium containing 10% FBS, and the medium was changed every 3 days. On the 7th day, the aggregates were recovered according to the method of Example 7 to detect changes in secretion factors of different groups of cells. The results are shown in FIG. Fig.21 As shown, when the amount of microspheres is too high (the ratio of microspheres to cells is 3:1), many microspheres cannot completely enter the aggregates and cannot form complete aggregates. It can be further seen from the quantitative statistical graphs (21B, C) that the actual ratios of hMSCs to microspheres before and after aggregate formation are 3.88:1 (the ratio of addition is 3:1), 6.19:1 (the ratio of addition is 5:1), 9.05:1 (the ratio of addition is 8:1), and 10.52:1 (the ratio of addition is 10:1), respectively, indicating that the amount of both added will affect the morphology of cell aggregates and the final ratio of the two in the aggregates. Fig.21 As shown in D, when the ratio of cells to microspheres is 5:1, the expression of stemness markers (Nanog and Sox-2), proliferation markers (Ki67) and paracrine factors (TGF-β, HGF and IDO) of the cell aggregates is the strongest. Therefore, in subsequent examples, cell aggregates were prepared according to the ratio of cells to microspheres of 5:1.

[0202] Example 15. Determination of the concentration of different subtypes of fusion proteins on the surface of P / Ch 2:1-IL-1β composite microspheres

[0203] hE-cad-Fc and hN-cad-Fc were diluted according to the concentrations of 5:1 (hE-cad-Fc: 16.6 μg / mL, hN-cad-Fc: 3.3 μg / mL), 3:1 (hE-cad-Fc: 15 μg / mL, hN-cad-Fc: 5 μg / mL), 1:1 (hE-cad-Fc: 10 μg / mL, hN-cad-Fc: 10 μg / mL), 1:3 (hE-cad-Fc: 15 μg / mL, hN-cad-Fc: 5 μg / mL), and 1:5 (hE-cad-Fc: 3.3 μg / mL, hN-cad-Fc: 16.6 μg / mL). The above solutions were used to prepare P / Ch 2:1 composite microspheres modified with different fusion proteins and 20 μg / mL collagen-modified P / Ch according to the method of Example 11. 2:1-IL-1β composite microspheres. Then, hMSCs and microspheres were mixed at a ratio of 5:1, and cell aggregates were prepared and cultured according to the method of Example 14. On the 5th day, RNA was extracted according to the method of Example 7, reverse transcription was performed, and the expression of related genes was detected. The results are shown in Fig. 22 As shown in the figure, when the ratio of hE-cad-Fc to hN-cad-Fc was 1:3, the expression of immune regulation-related markers (STC-1, TSG-6) in hMSCs aggregates was the strongest, and the expression of stemness markers (Sox-2) and paracrine factors (HGF, TGF-β) was higher than other ratios. Therefore, we concluded that the ratio of hE-cad-Fc: hN-cad-Fc to immobilize fusion proteins on the surface of composite microspheres was 1:3 for subsequent studies.

[0204] Example 16. Preparation and characterization of hMSCs aggregates mediated by fusion protein modified P / Ch 2:1-IL-1β composite microspheres

[0205] Fusion protein-modified P / Ch 2:1-IL-1β composite microspheres and 20 μg / mL collagen-modified P / Ch 2:1-IL-1β composite microspheres were prepared according to the ratio optimized in Example 14. Cell aggregates were prepared according to the optimized ratio in Example 15 and the method in Example 6, placed in a 6-well culture plate, and cultured in DMEM / F12 medium containing 10% FBS. The medium was changed every 3 days. Specifically, (1) cell aggregates containing only hMSCs but no microspheres, referred to as the control group; (2) cell aggregates containing only hE-cad-Fc modified P / Ch 2:1-IL-1β microspheres, referred to as the MEP group; (3) cell aggregates containing only hN-cad-Fc modified P / C-h2:1-IL-1β microspheres, referred to as the MNP group; (4) cell aggregates containing P / Ch 2:1-IL-1β microspheres modified with hE-cad-Fc and hN-cad-Fc in a ratio of 1:3, referred to as the ME / NP group; and (5) cell aggregates containing P / Ch 2:1-IL-1β microspheres modified with type I collagen, referred to as the collagen group. The prepared cell aggregates were observed by taking photos under a microscope, and then the cell aggregates were collected and washed twice with PBS buffer, and then fixed overnight in a special fixative for electron microscopy. After dehydration with freshly prepared gradient ethanol (100%, 95%, 90%, 80%, 70%, 60% for 10 minutes each), they were glued to conductive glue, sprayed with gold, and observed on a SEM. The results are shown in Fig.23 As shown in the figure, the prepared aggregates have complete structures and clear edges, and the microspheres inside the aggregates can be clearly observed. In addition, the diameters of the prepared different aggregates are basically the same, indicating that the fusion protein surface-modified microspheres have no significant effect on the formation and size of hMSCs aggregates.

[0206] Example 17. Effects of fusion protein matrix on paracrine and immune regulation functions of hMSCs aggregates

[0207] Different groups of cell aggregates were prepared according to the method in Example 14, and pre-cultured in DMEM / F12 medium containing IL-1β10 ng / mL and 10% FBS for 3 days. Total RNA was extracted according to the method in Example 7, and cDNA was obtained by reverse transcription. Using cDNA as a template, fluorescence quantitative PCR detection and data statistics were performed according to the method in Example 8. The primer sequences and annealing temperatures of related genes are shown in Table 5. The results are shown in Table 5. Fig.24As shown in the figure, the expression levels of HGF, IDO, IL-6, LIF and TGF-β in the aggregates of the ME / NP group were significantly higher than those in other control groups. hE / N-cad-Fc promoted the upregulation of genes related to paracrine factors in hMSCs aggregates, indicating that hE / N-cad-Fc plays a role in organogenesis, angiogenesis and immune response by promoting the paracrine function of hMSCs and improving hMSCs aggregates. Fig.25 It can be seen that the expression levels of three genes, TSG-6, STC-1 and PGE-2, in the ME / NP group were significantly higher than those in other control groups. hMSCs exert anti-inflammatory, anti-apoptotic and immunoregulatory effects by secreting related factors. This result indicates that the hE / N-cad-Fc co-modified microspheres have a significant effect on improving the anti-inflammatory and immunoregulatory functions of hMSCs aggregates.

[0208] Example 18. Study on the expression of endogenous cadherin in hMSCs aggregates mediated by fusion protein matrix

[0209] Different groups of cell aggregates were prepared according to the method in Example 14, and pre-cultured for 3 days in DMEM / F12 medium containing IL-1β10 ng / mL and 10% FBS. Total RNA was extracted according to the method in Example 7, and the expression levels of E-cadherin and N-cadherin genes of hMSCs were detected. Protein was extracted according to the method in Example 9, and the changes in protein levels were detected by WB. The results are shown in Figure 14. Fig.26 As shown, hE / N-cad-Fc can jointly enhance the expression of endogenous E-cadherin and N-cadherin in hMSCs aggregates. The up-regulation of endogenous cadherin in hMSCs, especially E-cadherin and N-cadherin, is closely related to its cell proliferation, migration, stemness maintenance, anti-apoptosis and paracrine ability. This indicates that cadherin fusion protein may further activate hMSCs related functions by enhancing the expression level of cell's own cadherin.

[0210] Example 19. Effect of fusion protein matrix-mediated hMSCs aggregates on macrophages

[0211] Macrophages in good condition were digested and counted on a cell counting plate according to 4×10 5 The cells were inoculated into the upper layer of a 24-well transwell chamber (pore size 0.4 μm) at a ratio of 10 cells / well. After the cells were completely attached to the wall, the culture medium was replaced with a culture medium containing 0.1 μg / mL LPS and placed in an incubator for stimulation for 90 min. After the stimulation, the LPS-containing culture medium was discarded and 2×10 5The density of hMSCs / well was added to the cell aggregates mediated by the fusion protein matrix in the lower layer of the chamber and placed in an incubator. After 5 hours of co-culture, the macrophage culture medium in the upper layer of the chamber was collected and centrifuged at 500×g for 10 minutes. The content of mTNFa was detected by Elisa according to the method in the manual. The upper layer macrophage protein was collected according to the method in Example 9, and the polarization of macrophages was detected during the WB detection process. The culture medium of the hMSCs aggregates in different groups in the lower layer was discarded and RNA was extracted according to the method in Example 7. The expression of hMSCs-related genes was detected during the detection process. The results are shown in Fig. 27 As shown, from Fig. 27 B shows that the ME / NP group aggregates can significantly reduce the secretion of TNF-α in mouse macrophages. Fig. 27 C and D show that the aggregates in the ME / NP group can promote more mouse macrophages to transform from M1 to M2. At the same time, the expression of hMSCs-related regulatory genes (PLA2, COX2 and PGEs) was the strongest in the ME / NP group, indicating that hE / N-cad-Fc promotes hMSCs to play an anti-inflammatory role in this process.

[0212] Example 20. Fusion protein matrix-mediated hMSCs aggregates improve the efficacy of hMSCs on colitis in mice

[0213] The mouse colitis model experiment was conducted with reference to the method of Matam Vijay-Kumar et al., using C57BL / 6J mice aged 6-8 weeks. Dextran sulfate sodium (DSS) was dissolved in sterile water to prepare a 3% DSS solution, and the drinking water of the mice was replaced with a 3% DSS solution every two days for 7 consecutive days, after which it was replaced with normal water. The specific steps are as follows:

[0214] 1. Take 20 healthy C57BL / 6J mice, weigh them, and randomly divide them into healthy group (Control), PBS group, M group and ME / NP group.

[0215] 2. The first day after the mice drank 3% DSS solution (recorded as day 0 of treatment), the cells were injected on the second day after the mice drank 3% DSS solution (recorded as day 1 of treatment), and the prepared cell aggregates of each group were resuspended in PBS buffer and injected into the abdominal cavity of the mice at a cell implantation amount of 1 million cells / mouse.

[0216] 3. Observe the condition of the mice every day, measure their weight, observe the morphology of their feces, and detect occult blood in their feces. Score the disease activity index of the mice according to the following table:

[0217] Table 12

[0218]

[0219]

[0220] 4. On the 14th day, the mice were killed by dislocating the neck, and the colon was taken out for photographing. After repeatedly flushing the intestine with 1640 culture medium, the intestine was placed in 4% paraformaldehyde for fixation for more than two weeks, and dehydrated, embedded, sliced ​​and H&E stained according to the method in Example 6 to observe the histological lesions of the mouse colon. The spleen of the mouse was removed, PBS buffer (containing 2% FBS) was added, and the mixture was fully ground and filtered (200 mesh filter), and the spleen cells were obtained by centrifugation at 1000g for 5 minutes at 4°C. 4 mL of red blood cell lysis buffer was added and incubated for 10 minutes, the supernatant was discarded by centrifugation, and the cells were resuspended in PBS buffer (containing 2% FBS) and counted. Single cells in the lymph nodes were obtained according to the same steps.

[0221] Take 1×10 6 Spleen cells were placed in a 96-well plate, the supernatant was discarded after centrifugation, 100 μL of cell stimulator (1:100 dilution) was added, and the cells were resuspended and incubated at 37°C for 4 hours. Wash once with PBS buffer (containing 2% FBS), add 100 μL of flow cytometry antibodies CD3 and CD8a (1:500 dilution in PBS buffer) to resuspend the cells, and incubate at 4°C in the dark for 30 minutes. After the incubation, wash once with PBS buffer containing 2% FBS, centrifuge at 1800×g for 5 minutes, add 100 μL of membrane permeabilization agent and incubate at 4°C for 20 minutes, wash once with PBS buffer, centrifuge at 2000×g for 5 minutes, add flow cytometry antibodies IFNr-PerCP, IL4 and IL17 (1:500 dilution in PBS buffer) respectively, mix well, incubate at 4°C in the dark for 30 minutes, wash once with PBS buffer, and detect by flow cytometry. Spleen cells were labeled with CD4-FITC and Foxp3 in the same way. The results are as follows Fig.28 As shown, the weight of mice in the PBS group showed a significant downward trend on the 5th day of treatment, which proved that the DSS enteritis model was successfully established in this process. The weight of mice in the ME / NP group began to recover on the 8th day, while the weight of mice in the M group did not recover until the 9th day. On the 14th day of the observation period, the weight of mice in the ME / NP group was significantly higher than that of the M group. At this time, the state of mice in the ME / NP group was similar to that of normal mice, with active emotions and normal diet. Fig.28 As can be seen in B, the ME / NP group had the lowest overall score based on the weight loss, fecal hardness, and fecal occult blood of the mice, proving that the disease in the ME / NP-treated group was the mildest, revealing that the hE / N-cad-Fc-modified microspheres have the effect of improving the immunoregulatory ability of hMSCs aggregates. Fig.28As shown in C, the colon of mice in the PBS group showed severe lesions and obvious necrosis, the colon was atrophied and shortened, and there was no feces with complete structure visible to the naked eye in the colon. However, the colon of mice in the ME / NP treatment group was complete in morphology, with complete granular feces inside the colon, and the colon length was closest to that of the normal group.

[0222] from Fig.29 As can be seen in A, the colon sections of normal mice have complete lamina propria, mucosa, submucosa and muscularis, a large number of epithelial cells and goblet cells, and basically no lymphocyte infiltration is observed. In the colon sections of mice in the PBS group, the colon epithelium was severely ulcerated, a large number of epithelial cells and goblet cells were lost, and a large number of lymphocyte infiltrations were present. This situation was slightly alleviated in the colons of mice in the M treatment group, but a large number of lymphocyte infiltrations were still observed, indicating that the M group aggregates had a slight inhibitory effect on the destruction of the colon epithelium. The colon structure of mice in the ME / NP treatment group was more complete, the degree of colon epithelial cell destruction was the lowest, the goblet cell loss was the smallest, and the lymphocyte infiltration was the least, indicating that the ME / NP aggregates effectively alleviated the colitis in mice, indicating that the hE / N-cad-Fc combined with the modified P / Ch 2:1-IL-1β composite microspheres significantly improved the anti-inflammatory and immune regulation functions of hMSCs aggregates.

[0223] from Fig.30 As can be seen in A, a large number of CD4 + and CD8 + The injection of ME / NP group aggregates effectively inhibited the CD4 + and CD8 + T cell infiltration. Fig.30 As can be seen in D, hMSCs in the ME / NP group can inhibit the Th 1 and Th 17 differentiation of T cells to the greatest extent, and the same conclusion can be drawn in the mesenteric lymph nodes. Therefore, the results show that the hMSCs aggregates mediated by hE / N-cad-Fc combined with modified P / Ch 2:1-IL-1β composite microspheres (ME / NP group) have higher immunomodulatory activity and inhibit the proliferation and differentiation of T cells into pro-inflammatory Th1 and Th17 cells.

[0224] Example 21. Sustained release of TGFβ1 by fusion protein surface modified P / Ch 2:1-TGFβ1 composite microspheres

[0225] According to the method in Example 4, P / Ch 2:1-TGFβ1 composite microspheres modified with fusion protein loaded with TGFβ1 (TGFβ1, Peprotech, USA) were prepared, and the release performance of TGFβ1 of P / Ch 2:1-TGFβ1 microspheres was tested according to the method in Example 4. The results are shown in FIG. Fig.31As shown, TGFβ1 factor in P / C-h2:1 composite microspheres was gradually released in the first 12 days, and the factor was continuously released in the next 15 days.

[0226] Example 22. Optimization of the ratio of hE-cad-Fc and hN-cad-Fc fusion proteins and their effects on endogenous N-cadherin expression in hMSCs aggregates

[0227] hE-cad-Fc and hN-cad-Fc were prepared into 10 μg / mL protein solutions. 1.5 mL of 10 μg / mL collagen solution, hE-cad-Fc and hN-cad-Fc in a ratio of 1:1 (hE-cad-Fc: 5 μg / mL, hN-cad-Fc: 5 μg / mL), 1:2 (hE-cad-Fc: 3.3 μg / mL, hN-cad-Fc: 6.6 μg / mL), and 1:3 (hE-cad-Fc: 2.5 μg / mL, hN-cad-Fc: 7.5 μg / mL) and a protein solution containing only 10 μg / mL hN-cad-Fc were added to a six-well PS culture plate. The six-well plate was placed in a cell culture incubator at 37°C for 2 h, and then the supernatant was discarded and washed three times with PBS buffer.

[0228] Then the cell density was 1×10 5 hMSCs were seeded onto six-well PS culture plates incubated with the above-mentioned protein mixtures of different subtypes and concentrations, and cartilage-directed differentiation induction medium (DF12 medium containing 5% FBS, 10 ng / mL TGFβ1, 200 nM 2-phospho-ascorbic acid, and 100 nM dexamethasone) was added and cultured in a cell culture incubator. After culturing for 1 week, the medium was discarded, and the cells were washed 3 times with PBS buffer and photographed. Fig.32 As shown in the figure, after one week of chondrogenic differentiation culture, all groups except the collagen group showed different degrees of cell aggregation. When the concentration ratio of hE-cad-Fc and hN-cad-Fc fusion proteins was 1:2, the cell aggregates generated by spontaneous aggregation were more uniform in size and higher in density, which was more conducive to the formation of the high cell density required for hMSCs to differentiate into chondrocytes.

[0229] According to the method in Example 11, a 20 μg / mL collagen solution, a hE-cad-Fc and hN-cad-Fc ratio of 1:1 (hE-cad-Fc: 10 μg / mL, hN-cad-Fc: 10 μg / mL), 1:2 (hE-cad-Fc: 6.5 μg / mL, hN-cad-Fc: 13 μg / mL) and 1:3 (hE-cad-Fc: 5 μg / mL, hN-cad-Fc: 15 μg / mL) and a 20 μg / mL hN-cad-Fc protein solution were prepared, and the above solutions were used to prepare P / Ch 2:1-TGFβ1 composite microspheres with different surface modifications. According to the method in Example 6, 9×106 cells and P / Ch 2:1-TGFβ1 composite microspheres modified with different proteins were mixed in a ratio of 1:3 to prepare cell aggregates. According to the method in Example 6, 3D culture was performed using alginate hydrogel. The differentiation was induced in the cartilage differentiation medium for one week, and the medium was changed every two days. On the seventh day, the alginate hydrogel spheres were lysed according to the method in Example 6, and the cell aggregates were recovered. mRNA was extracted according to the method in Example 7 for RT-PCR, and the primers used are shown in Table 13. Protein was extracted according to the method in Example 9 for WB detection. The experimental results are shown in Fig.33 As shown, when the ratio of hE-cad-Fc and hN-cad-Fc was 1:2, the expression of Sox9, type 2 collagen and endogenous E-cadherin were significantly higher than those in other groups, both at the gene level and the protein level. This proves that when the ratio of hE-cad-Fc and hN-cad-Fc was 1:2, the combined modified P / C-h2:1-TGFβ1 composite microspheres could better promote hMSCs to express chondrocyte marker proteins while maintaining high expression of E-cadherin.

[0230] Collagen solution (20 μg / mL), hN-cad-Fc (20 μg / mL), hE-cad-Fc and hN-cad-Fc (1:2, hE-cad-Fc: 6.5 μg / mL, hN-cad-Fc: 13 μg / mL) were used to incubate P / C-h2:1-TGFβ1 composite microspheres, respectively. According to the method of Example 6, the P / C-h2:1-TGFβ1 composite microspheres modified with different protein solutions and hMSCs were prepared into cell aggregates, which were recorded as hMSCs aggregates containing collagen-modified P / C-h2:1-TGFβ1 composite microspheres (MCP), hMSCs aggregates containing hN-cad-Fc-modified P / C-h2:1-TGFβ1 composite microspheres (MNP), and hMSCs aggregates containing hE-cad-Fc and hN-cad-Fc proteins modified in a ratio of 1:2 (MENP). In addition, pure cell aggregates (M) without P / Ch 2:1-TGFβ1 composite microspheres were prepared as a control group. The cells were cultured in cartilage induction differentiation medium (M group: DF12 + 5% FBS + 10 ng / mL TGFβ1 + 200 nM 2-p-asc + 100 nM Dex, and the aggregate group containing P / C-h2:1-TGFβ1 composite microspheres: DF12 + 5% FBS + 200 nM 2-p-asc + 100 nM Dex) for 7 consecutive days, with the medium changed every two days. mRNA was extracted according to the method of Example 7 for RT-PCR detection, and the relevant primers are shown in Table 13. Protein was extracted according to the method of Example 9, and the expression of endogenous N-cadherin in cells was detected over time. The results are shown in Table 13. Fig.34 As shown in the figure, on the third day of differentiation, the gene and protein expression levels of N-cadherin in the MENP group reached a peak, and in the subsequent differentiation process, the gene and protein expression of N-cadherin decreased sharply; while the N-cadherin gene and protein expressed by cells in the M group reached a peak on the fifth day. And the peak values ​​of N-cadherin in the M, MCP, and MNP groups were all lower than those in the MENP group. This shows that the combination of hE-cad-Fc / hN-cad-Fc (1:2) in MENP can accelerate the upregulation of N-cadherin expression in hMSCs, promote the process of interstitial condensation of hMSCs, and improve the directional differentiation process of hMSCs into chondrocytes.

[0231] Table 13

[0232]

[0233] Example 23. hE / N-cadherin-Fc combined with modified P / Ch-TGFβ1 microspheres promotes hMSCs aggregates to differentiate into cartilage in vitro

[0234] Different groups of hMSCs aggregates were prepared according to the method of Example 22, and chondrogenic differentiation was carried out in alginate hydrogel for 4 weeks, and samples were taken every 7 days. mRNA was extracted according to the method in Example 7 for RT-PCR, and the relevant primers are shown in Table 13. Protein was extracted according to the method in Example 9 for WB detection. The antibodies used are as follows: Sox9 (Abcam, USA), proteoglycan (Abcam, USA), type 2 collagen (Abcam, USA), and β-actin (Abcam, USA). After directional induction of differentiation for 1 week, 2 weeks, 3 weeks, and 4 weeks, the expression of genes and proteins of Sox9, type 2 collagen, and proteoglycan in different groups of aggregates is as follows: Fig.35 As shown. After 1 week of differentiation, the expression of proteoglycans in the MENP group was significantly higher than that in the other two groups; after 2 weeks of differentiation, the expression of Sox9, type 2 collagen and proteoglycans in the MENP group were significantly higher than those in the M, MCP and MNP groups; after 3 and 4 weeks of differentiation, the expression of proteoglycans in the MENP group was still significantly higher than that in the other three groups. This shows that after the surface of the P / C-h2:1-TGFβ1 composite microspheres was modified with hE-cad-Fc / hN-cad-Fc (1:2), the expression of chondrocyte-related marker proteins including Sox9, type 2 collagen and proteoglycans can be continuously enhanced, and it also shows that it can accelerate the process of hMSCs differentiation into chondrocytes.

[0235] According to the method of Example 22, hMSCs aggregates of different compositions were prepared, and cartilage differentiation was induced and cultured in alginate hydrogels for 4 weeks. Samples were taken every 2 weeks, and the aggregates were recovered according to the method of Example 6, fixed in 4% paraformaldehyde for more than 2 days, embedded in paraffin, and stained with Alcian blue (Solebol) after sectioning. The specific process of Alcian blue staining is as follows:

[0236] 1. Bake the slides at 60℃ until the paraffin melts, and then soak them in xylene I and xylene II for 5 min each to dewax;

[0237] 2. Soak in 100%, 95%, 90%, 80%, and 70% alcohol gradient for 5 minutes each;

[0238] 3. Rinse with distilled water for 5 minutes and incubate in acidified solution for 3 minutes;

[0239] 4. Stain with Alcian staining solution for 30 minutes, rinse with running water for 5 minutes;

[0240] 5. Soak in 70%, 80%, 90%, and 100% alcohol for 5 seconds each to decolorize;

[0241] 6. Xylene I and xylene II were transparentized for 5 minutes each;

[0242] 7. Seal the slide with neutral gum and observe under an upright microscope.

[0243] like Fig.36 As shown, after 2 weeks of differentiation, the P / Ch 2:1-TGFβ1 composite microspheres in the MCP, MNP and MENP groups were all degraded, and the Alcian blue staining in the MENP group was the strongest; after 4 weeks of differentiation, the P / Ch 2:1-TGFβ1 composite microspheres in the MCP, MNP and MENP groups were basically completely degraded, and the Alcian blue staining effect in the MENP group was still the strongest. This indicates that hE-cad-Fc / hN-cad-Fc (1:2) in the MENP group can promote the differentiation of hMSCs into chondrocytes, and the chondrocytes differentiated in the MENP group can maintain the functional characteristics of chondrocytes for a long period of time.

[0244] Example 24. hE / N-cadherin-Fc combined with modified P / Ch-TGFβ1 composite microspheres mediated hMSCs aggregates inhibit calcification in the late stage of chondrogenic differentiation

[0245] According to the method of Example 22, different groups of hMSCs aggregates were prepared and cultured for cartilage differentiation in alginate hydrogel for 4 weeks. According to the method of Example 6, they were fixed with 4% paraformaldehyde for more than 2 days, embedded in paraffin and sectioned, and then stained with safranin fast green (Solabol). The specific process is as follows:

[0246] 1. Bake the slides at 60℃ until the paraffin melts, and then soak them in xylene I and xylene II for 5 min each to dewax;

[0247] 2. Soak in 100%, 95%, 90%, 80%, and 70% alcohol gradient for 5 minutes each;

[0248] 3. Rinse with distilled water for 5 minutes, add Weigert staining solution, and stain for 3 minutes;

[0249] 4. Differentiate in acidic ethanol differentiation solution for 15 seconds;

[0250] 5. Wash with distilled water for 5 minutes and dye with fast green dye for 7 minutes;

[0251] 6. Wash with distilled water for 5 minutes and stain with Safranin O stain solution for 3 minutes;

[0252] 7. Wash with distilled water for 5 minutes, wash with acetic acid solution for 1 minute, and wash with distilled water for 5 minutes;

[0253] 8. Soak in 70%, 80%, 90%, and 100% alcohol for 5 seconds each to decolorize;

[0254] 9. Xylene I and Xylene II were transparentized for 5 minutes each;

[0255] 10. Seal the slides with neutral gum and observe under an upright microscope.

[0256] The results are as follows Fig.37 As shown, after 2 weeks of differentiation, compared with the M, MCP and MNP groups, the MENP group had the strongest safranin staining effect, and none of the four groups had obvious fast green staining; after 4 weeks of differentiation, the cells in the M group began to show a loose morphology; while the MCP, MNP and MENP groups containing P / Ch 2:1-TGFβ1 composite microspheres were still able to maintain the aggregate state, the MCP group showed obvious fast green staining, the MNP group had slight fast green staining, the MENP group did not show fast green staining and still maintained a strong safranin staining effect. This shows that after 4 weeks of differentiation, the cells in the M group died and fell off, the cells in the MCP and MNP groups showed different degrees of calcification, and the MENP group was still able to maintain the characteristics of chondrocytes well without calcification.

[0257] Different groups of hMSCs aggregates were prepared according to the method of Example 22, and chondrogenic differentiation cultured in alginate hydrogel for 4 weeks. RNA was extracted according to the method of Example 7, and PCR was performed to detect the gene expression of MMP13, ALP, type 10 collagen and type 2 collagen. The ratio of the gene expression levels of type 2 collagen and type 10 collagen was statistically analyzed. The primer sequences used are shown in Table 14.

[0258] Table 14

[0259]

[0260] like Fig.38 As shown in the figure, after 4 weeks of directional induction of chondrocyte differentiation, compared with the MNP and MENP groups, the M and MCP groups had higher expression levels of MMP13, ALP and type 10 collagen, and a lower ratio of type 2 collagen to type 10 collagen, indicating that the M and MCP groups had stronger cartilage calcification, while the expression levels of MMP13, ALP and type 10 collagen in the MENP group were significantly reduced, indicating that the chondrocytes differentiated from this group were more stable, and the combined modification of hE-cad-Fc / hN-cad-Fc (1:2) can better inhibit the calcification of differentiated mature chondrocytes.

[0261] Example 25. Regulatory mechanism of hE / N-cadherin-Fc co-modified P / Ch-TGFβ1 microspheres mediated in vitro differentiation of hMSCs aggregates into chondrocytes

[0262] Different groups of hMSCs aggregates were prepared according to the method of Example 22, and cartilage induction differentiation was cultured in alginate hydrogel for 4 weeks. The aggregates were recovered according to the method in Example 6, fixed with 4% paraformaldehyde for more than 2 days, and washed three times with PBS buffer for 5 minutes each time. Then 100 μL of goat serum (Biyuntian) (diluted with PBS buffer 1:200) was added to each group of aggregates, and blocked at room temperature for 30 minutes. Then β-catenin antibody (1:500, Abcam) and E-cadherin antibody (1:500, Abcam) were diluted with PBS buffer, and 50 μL of each was added to each group of aggregates. After incubation at 4°C overnight, the PBS buffer was washed 3 times for 5 minutes each time. FITC-labeled goat anti-mouse IgG antibody (Invitrogen, USA, Catalog No. A-10680) and Rhodamine-labeled goat anti-rabbit secondary antibody (Invitrogen, USA, Catalog No. 35560) were diluted 1:500 with PBS buffer, and 50 μL of each was added to each group of aggregates, incubated at room temperature for 2 h, and washed 3 times with PBS buffer, each time for 5 min. Then 20 μL of anti-fluorescence quencher containing DAPI (Southern Biotech, USA, Catalog No. 0100-20) was added to each group of aggregates, and photographed using a laser confocal microscope.

[0263] According to the method of Example 22, different groups of hMSCs aggregates were prepared, and cartilage induction differentiation was cultured in alginate hydrogel for 4 weeks. The aggregates were recovered according to the method in Example 6, and then the aggregates were placed in a 1.5mL EP tube, 500μL of 0.25% trypsin was added, and 500μL of DF12 medium containing 10% FBS was added to terminate the digestion after shaking in a 37°C water bath for 5min. The supernatant was discarded and resuspended with PBS buffer, and centrifuged at 4°C 1000×g for 5min. Then, the proteins in the nucleus and cytoplasm were extracted respectively using a cell nuclear extraction kit (Solarbio, USA, item number R0050), and the nuclear and cytoplasmic proteins were separated and then subjected to WB detection according to the method of Example 9. The main steps of nuclear and cytoplasmic protein separation are as follows:

[0264] 1. Wash single cells with PBS buffer, centrifuge at 500g for 5 minutes, and collect cells;

[0265] 2. Add 200 μL of plasma protein extraction reagent to every 20 μL of cell pellet, vortex thoroughly for more than 15 seconds to completely disperse the cells, place on ice for 10 minutes, and centrifuge at 12,000 × g for 10 minutes at 4°C;

[0266] 3. Collect the cell nuclear precipitate at the bottom of the centrifuge tube and collect the cytoplasmic protein solution in the supernatant for subsequent use;

[0267] 4. After aspirating the supernatant, add 50 μL of nuclear protein extraction reagent to the cell nuclear pellet;

[0268] 5. Pipette or vortex for more than 15 seconds until completely dispersed, ice bath for 10 minutes, 4°C, and centrifuge at 12000g for 10 minutes;

[0269] 6. Aspirate the supernatant into a pre-cooled sample tube, which is the nuclear protein and should be stored at -80℃.

[0270] The results are as follows Fig.39 As shown in the figure, the expression of E-cadherin in the MENP group was significantly higher than that in the other three groups, and the β-catenin in the MENP group was mainly located in the cytoplasm; the expression of E-cadherin in the M, MCP and MNP groups was less, and β-catenin was mainly located in the nucleus, indicating that the combined modification of hE-cad-Fc / hN-cad-Fc (1:2) in the MENP group can effectively promote the differentiation of hMSCs into chondrocytes and promote the expression of the cell's own E-cadherin, so that β-catenin is more located in the cytoplasm and inhibits its entry into the nucleus to induce chondrocyte ossification.

[0271] According to the method of Example 22, different groups of hMSCs aggregates were prepared, and chondrogenic differentiation was cultured in alginate hydrogels for 4 weeks. The aggregates were recovered according to the method in Example 6, fixed with 4% paraformaldehyde for more than 2 days, blocked according to the method in the above example, incubated with YAP antibody (1:500 dilution in PBS buffer, Abcam) and E-cadherin antibody (1:500 dilution in PBS buffer, Abcam), washed, incubated with FITC-labeled goat anti-mouse IgG antibody and Rhodamine-labeled goat anti-rabbit secondary antibody, and then 20 μL of anti-fluorescence quencher containing DAPI was added, and photographed using a laser confocal microscope. The results are shown in FIG. Fig.40 A.

[0272] hMSCs aggregates of different compositions were prepared according to the method of Example 22, and chondrogenic differentiation was cultured in alginate hydrogel spheres for 4 weeks. Nuclear and cytoplasmic proteins were separated according to the above steps, and WB detection was performed according to the method of Example 9. The results are shown in Fig.40B, The total expression of YAP in the MENP group was significantly lower than that in the M, MCP and MNP groups, and the expression of E-cadherin in the MENP group was significantly higher than that in the M, MCP and MNP groups. In the MCP group, the phosphorylated YAP in the cytoplasm was the least, and the YAP in the nucleus was the most; in the MENP group, the phosphorylated YAP in the cytoplasm was the most, and the YAP in the nucleus was the least. This indicates that YAP in the MENP group is more phosphorylated and more located in the cytoplasm, inhibiting its entry into the nucleus to induce chondrocyte calcification; while in the M, MNP and MCP groups, YAP is more located in the nucleus, and will bind to DNA to promote chondrocyte proliferation, causing cartilage hypertrophy and calcification.

[0273] Example 26. Study on the treatment of rat knee cartilage injury by hE / N-cadherin-Fc combined with modified P / Ch-TGFβ1 microspheres mediated by hMSCs aggregates

[0274] 72 female SD rats (11 weeks, 320-340 g) were purchased from Nanjing Qinglongshan Animal Farm for this in vivo experiment. After general anesthesia, 60 rats were randomly selected to make cartilage defect models (2.5 mm in diameter and 1.5 mm in height) at the femoral groove of the knee joint. The other 12 rats were directly sutured without modeling treatment and served as the control group. Then, the M, MCP, MNP and MENP aggregates prepared in Example 22 (650 cell aggregates, about 4×10 5 Cells were injected into the joint cavity of cartilage-defective rats, and an equal volume of PBS buffer was injected as a blank control. Six parallels were set up in each group. Gross observation, histology and MicroCT analysis were performed at 3 and 6 weeks.

[0275] At 3 and 6 weeks after surgery, the femurs of SD rats were removed for gross observation. Fig.41 As shown, at the 3rd week after surgery, compared with the PBS group, the depression marks at the cartilage defect sites in the M, MCP and MENP groups were significantly reduced, and a small amount of hyaline cartilage layer appeared on the surface of the defect; at the 6th week after surgery, the boundary marks at the cartilage defect sites in the MENP group disappeared, and the defect sites were covered with uniform hyaline cartilage tissue; while the depression marks at the defects in the PBS, M and MCP groups were still significant.

[0276] The femur was fixed with 4% paraformaldehyde for more than two days, and then decalcified with 10 mL of 10% EDTA decalcification solution. The decalcification solution was changed every two days for 4 weeks. Paraffin embedding and sectioning were performed according to the method in Example 6, and safranin fast green histological staining was performed according to the method in Example 24. The results are as follows: Fig.42As shown, at the third week after surgery, the defect depth in the PBS group was the deepest, and the filled tissue was negative for safranin staining; the defect site in the M group was filled with fibrous tissue, with irregular tissue orientation and an uneven surface; the defect site in the MCP group was filled with a small amount of cells, with negative safranin staining, and the surface of the filled tissue was uneven; the surface of the MNP tissue was filled with uniform thickness and a smoother surface, but the safranin staining result of the injured site was negative; the thickness of the filled tissue in the MENP group was close to that of the natural cartilage tissue, with a smooth surface, densely filled cells, and a small amount of safranin-positive tissue filled in the injured site. At the sixth week after surgery, the PBS group still had obvious traces of cartilage defects; the tissue filled in the defect site in the M group was severely fibrotic, with obvious surface defects and obvious gaps between tissues; the defect site in the MCP group was still obviously concave, and there were large gaps between cells in the defect site; the tissue filled in the defect site in the MNP group was denser, with some safranin-positive tissue, but the filling thickness was thicker than that of the natural cartilage tissue; the thickness of the new tissue formed in the defect site in the MENP group was close to that of the natural cartilage tissue, with safranin-positive tissue and a uniform surface. This showed that 6 weeks after surgery, the MENP group had more cartilage tissue in the filling tissue of the defect site and a better repair effect.

[0277] Example 27. Study on the treatment of subchondral bone defects in rats with hE / N-cadherin-Fc combined with modified P / Ch microspheres mediated by hMSCs aggregates

[0278] The femurs of the SD rats at 3 and 6 weeks after the operation in Example 26 were removed and analyzed by MicroCT (SCANCO MEDICALvivaCT 80, Switzerland). The results are as follows: Fig.43 As shown, from Fig.43 Aa and 43Ab showed that at the third week after surgery, the depth of the subchondral bone defect in the MENP group was significantly shallower than that in the PBS group, and the defect site had different degrees of bone filling; the subchondral bone in the M group, MCP group, and MNP group still had obvious subchondral bone defect marks, and the defect depth was not effectively repaired compared with the PBS group. Fig.43 Ac and Fig.43 AdIt can be seen that the BV / TV values ​​of the MNP group and the MENP group were significantly higher than those of the M group; the Tb / Th values ​​of the MNP group and the MENP group were also significantly higher than those of the M group. This indicates that at 3 weeks after surgery, the comprehensive degree of subchondral bone repair in the MENP group was better than that in the MNP group, and was better than that in the PBS group, the M group, and the MCP group.

[0279] from Fig.43Ba and 43Bb show that at week 6 after surgery, compared with the PBS group and the M group, the depth of the defect in the subchondral bone in the MENP group was significantly shallower, and the depression marks at the defect site were almost filled with newly formed bone tissue, and there were no obvious depression marks; the subchondral bone in the M group, the MCP group, and the MNP group still had obvious subchondral bone defect marks, and the defect depth was not effectively repaired compared with the PBS group. At the same time, the proliferation of the defect in the M group was more serious than that in the other groups. Fig.43 Bc and Fig.43 Bd shows that the BV / TV values ​​of the MNP group and the MENP group are significantly higher than that of the M group, and the BV / TV value of the MNP group is also significantly higher than that of the MNP group; the Tb / Th value of the MNP group and the MENP group is also significantly higher than that of the M group. This shows that at 6 weeks after surgery, the MENP group has a better degree of repair of subchondral bone than the MNP group, and is more significantly better than the PBS group, the M group and the MCP group.

[0280] Example 28. Mechanism of repair of rat articular cartilage defects by hE / N-cadherin-Fc co-modified P / Ch microspheres mediated by hMSCs aggregates

[0281] The 3- and 6-week cartilage samples were fixed and decalcified according to the method in Example 26, and then embedded and sliced ​​according to the method in Example 6. Then, immunostaining of Ku80 protein and type II collagen was performed. A 0.4% pepsin solution was prepared with 0.1M hydrochloric acid, dripped on the cartilage sample, and incubated at 37°C for 30 minutes for antigen repair. Blocking was performed according to the method in Example 25, and Ku80 antibody (1:500 dilution in PBS buffer, Abcam) and type 2 collagen antibody (1:500 dilution in PBS buffer, Abcam) were incubated, washed, and incubated with FITC-labeled goat anti-mouse IgG antibody and Rhodamine-labeled goat anti-rabbit secondary antibody. Then, 20 μL of anti-fluorescence quencher containing DAPI was added, and photos were taken using a laser confocal microscope. The results are as follows. Fig.44 As shown, 3 weeks after in vivo injection treatment, compared with the M, MCP and MNP groups, the MENP group had more Ku80 staining, indicating more positive human cells, and more type 2 collagen was expressed around them, indicating that the aggregates in the MENP group could better locate to the cartilage defect site, survive longer in vivo, differentiate into more mature chondrocytes, express type 2 collagen in the cartilage extracellular matrix, and fill the cartilage defect site in situ.

[0282] hMSCs aggregates of different compositions were prepared according to the method of Example 22 and cultured in 3D in alginate hydrogel. 10 ng / mL IL-1β was added to the chondrogenic differentiation medium (M group: DF12+5% FBS+10 ng / mL TGFβ1+200 nM 2-p-asc+100 nM Dex, aggregate group containing P / Ch 2:1-TGFβ1 composite microspheres: DF12+5% FBS+200 nM 2-p-asc+100 nM Dex) to simulate the arthritis microenvironment in vitro, and the medium was changed every 24 h. After 48 h of induction, the aggregates were recovered according to the method in Example 6, and RNA was extracted according to the method in Example 7 for RT-PCR to detect the gene expression of TGFβ, IL-10 and IL-4. The primer sequences used are shown in the following table.

[0283] Table 15

[0284]

[0285] The results are as follows Fig.45 As shown, after 48 hours of induction of the in vitro arthritis microenvironment, the gene expression of three anti-inflammatory factors, IL-4, IL-10 and TGFβ, in the MENP group aggregates was significantly higher than that in the M group, and the IL-10 gene expression in the MENP group was significantly different from that in the MNP group, indicating that the MENP aggregates had stronger gene expression of anti-inflammatory factors IL-4, IL-10 and TGFβ in the arthritis microenvironment. When repairing the articular cartilage defect model, the MENP aggregates can respond to the inflammatory response of the injury site early, secrete more anti-inflammatory factors, and reduce the inflammatory response of the injury site.

Claims

1. A modified matrix comprising an E-cadherin-Fc fusion protein and a N-cadherin-Fc fusion protein, wherein the matrix is ​​a hydrophobic microsphere; in, The matrix is ​​PLGA / chitosan-heparin core-shell composite microspheres.

2. The modified matrix according to claim 1, wherein The E-cadherin is human E-cadherin, and Fc is Fc of IgG; the N-cadherin is human N-cadherin, and Fc is Fc of IgG.

3. The modified matrix according to claim 1, wherein The Fc is the Fc of IgG1.

4. A method for preparing a modified matrix, comprising modifying the matrix by mixing an E-cadherin-Fc fusion protein and a N-cadherin-Fc fusion protein with the matrix; in, The matrix is ​​hydrophobic microspheres, and is PLGA / chitosan-heparin core-shell structure composite microspheres.

5. A cell aggregate, which is an aggregate formed by the modified matrix according to any one of claims 1 to 3 and cells; The cells are mesenchymal stem cells. The cell aggregate according to claim 5 , wherein the cells are derived from mammals. The cell aggregate according to claim 5 , wherein the cells are derived from humans, pigs or mice.

8. The cell aggregate of any one of claims 5-7, wherein the ratio of cells to modified matrix is ​​3:1 to 10:

1.

9. The cell aggregate according to any one of claims 5-7, wherein the ratio of cells to modified matrix is ​​3:1 or 5:

1.

10. Use of the cell aggregate according to any one of claims 5 to 9 in the preparation of a medicament for treating the following diseases: colitis, arthritis and acute and chronic articular cartilage defects.

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