CN116271241A - Modified asymmetric SIS membrane for tissue repair, preparation method and application thereof - Google Patents
Modified asymmetric SIS membrane for tissue repair, preparation method and application thereof Download PDFInfo
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- CN116271241A CN116271241A CN202111497975.6A CN202111497975A CN116271241A CN 116271241 A CN116271241 A CN 116271241A CN 202111497975 A CN202111497975 A CN 202111497975A CN 116271241 A CN116271241 A CN 116271241A
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Abstract
The present disclosure relates to a modified asymmetric SIS membrane for tissue repair, a method of preparing the same, and applications thereof. In particular, the present disclosure relates to compositions containing fusion polypeptide modified asymmetric SIS membranes and exosomes, as well as methods of making and using the compositions. The present disclosure demonstrates that fusion polypeptide-mediated asymmetric SIS membranes and compositions comprising the foregoing asymmetric SIS membranes and exosomes have excellent biological functions, significantly increased loading rates of exosomes on asymmetric SIS membranes, provide suitable microenvironments for healing, remodeling of bone tissue, and have significantly increased ability to induce bone regeneration.
Description
Technical Field
The present disclosure belongs to the field of biomedical materials, and in particular relates to a modified asymmetric SIS membrane for tissue repair, a preparation method and applications thereof.
Background
Diseases such as wounds, inflammation and tumors are common causes of tissue damage, and repair of tissue damage requires provision of a microenvironment suitable for cell proliferation and migration. For bone tissue repair, guided bone regeneration (guided bone regeneration, GBR) techniques based on barrier membranes are widely used. GBR techniques employ a barrier membrane (also known as GBR membrane) to cover the bone defect, exclude interference from non-osteogenic tissue during the bone healing process, and at the same time provide a favorable environment for bone regeneration to promote regeneration of bone tissue.
GBR membranes are one of the main factors determining the application effect of GBR technology, and currently, GBR membranes can be classified into absorbable GBR membranes and non-absorbable GBR membranes. The non-absorbable GBR membrane is made of polytetrafluoroethylene, titanium and other materials, and needs to be taken out through secondary operation, so that the pain and the cost of a patient are high. The absorbable GBR membrane is made of absorbable materials such as collagen and polylactic acid, and is widely used because of convenient use and no need of secondary surgical excision. Currently, absorbable Bio-Gide membranes are a biomimetic layered membrane with an asymmetric structure that acts as a barrier towards the dense layer of soft tissue to prevent fibroblast invasion into bone defects; the porous layer facing the bone defect has high porosity, and can promote the adhesion of osteoblasts and stabilize thrombus. At present, bio-Gide membrane has been applied clinically and has achieved positive therapeutic effects. However, bio-Gide membranes lack bioactive substances, which have insufficient ability to induce bone regeneration.
Extracellular matrix (ECM) materials are good matrices for tissue repair reconstruction; due to its acellular biological network, it has become an important scaffold material for tissue engineering technology in recent years. Small Intestinal Submucosa (SIS) is an ECM material with excellent properties, low immunogenicity, excellent mechanical properties, and tissue regeneration capability. Therefore, SIS membranes are widely used for repair of damaged tissues such as skin, cardiovascular, abdominal wall, cartilage defects, and the like, and satisfactory results are obtained.
By combining exosomes with SIS membranes, SIS materials with good tissue inducing properties can be obtained. However, due to the dense texture of SIS membranes, it is difficult to provide adequate growth space for bone cell growth, and only a few studies have been conducted to use SIS materials as GBR membranes. Therefore, how to realize the material modification of SIS films is an important problem to be solved at present.
Disclosure of Invention
Problems to be solved by the invention
In view of the problems existing in the prior art, for example, the loading of exosomes to SIS membranes has the defects of low loading efficiency, abrupt release in vivo, structural damage of exosomes and the like, and the SIS membranes have difficulty in providing a suitable growth space for migration and proliferation of bone cells. Therefore, the present disclosure provides an asymmetric SIS membrane, wherein a dense layer of the asymmetric SIS membrane can effectively isolate soft tissues, and a porous structure of the porous layer can provide a proper three-dimensional space for adhesion and proliferation of bone cells, and a stable growth space for proliferation and growth of the bone cells. In addition, due to the high porosity of the porous layer and the mediation of the fusion polypeptide, exosomes can be stably and efficiently loaded on the asymmetric SIS membrane, a proper microenvironment is provided for healing and remodeling of bone tissues, and the capability of inducing bone regeneration is remarkably improved.
Solution for solving the problem
(1) A composition, wherein the composition comprises a fusion polypeptide modified asymmetric SIS membrane and exosomes; the asymmetric SIS membrane has oppositely facing porous and dense layers with at least a portion of the exosomes being supported inside and/or on the surface of the porous layer.
(2) The composition of (1), wherein the sequence of the fusion polypeptide comprises at least one of the group consisting of the sequences shown in seq id no:
(i) Consists of the sequence as shown in SEQ ID NO:5 and the sequence shown as SEQ ID NO:6, a group consisting of the sequences shown in fig. 6;
(ii) Sequences in which there is one, two, three, four or five conservative substitutions compared to the sequences shown in (i);
preferably, the sequence of the fusion polypeptide consists of at least one of the group consisting of:
(i) Consists of the sequence as shown in SEQ ID NO:5 and the sequence shown as SEQ ID NO:6, a group consisting of the sequences shown in fig. 6;
(ii) There is one, two, three, four or five conservatively substituted sequences compared to the sequence set forth in (i).
(3) The composition of (1) or (2), wherein both ends of the fusion polypeptide bind the exosomes and the asymmetric SIS membrane, respectively;
preferably, the exosomes are derived from mesenchymal stem cells; more preferably, the exosomes are derived from exosomes of bone marrow mesenchymal stem cells.
(4) The composition according to any one of (1) to (3), wherein the asymmetric SIS film is obtained by partially immersing a SIS film in liquid nitrogen, a portion of the SIS film immersed in liquid nitrogen forms the dense layer, and a portion of the SIS film not immersed in liquid nitrogen forms the porous layer;
alternatively, the asymmetric SIS membrane is obtained by partial immersion of the SIS membrane in dry form in liquid nitrogen;
preferably, the dense and porous layers of the asymmetric SIS membrane are each loaded with exosomes.
(5) The composition of any one of (1) - (4), wherein the composition is obtained by incubating the asymmetric SIS membrane with the fusion polypeptide and the exosome.
(6) A method for producing the composition according to any one of (1) to (5), wherein the method comprises the following (a) to (d) or (i) to (iv):
(a) Dissolving the fusion polypeptide into a solvent to obtain a solution containing the fusion polypeptide;
(b) Mixing and incubating the solution obtained in the step (a) and the exosome to obtain an incubation solution;
(c) Applying the incubation solution from step (b) to at least one side of the porous layer of the asymmetric SIS membrane; preferably, the asymmetric SIS membrane is immersed in the incubation solution obtained in step (b);
(d) Drying the asymmetric SIS film with the incubation solution on the surface to obtain the composition; or alternatively
(i) Dissolving the fusion polypeptide into a solvent to obtain a solution containing the fusion polypeptide;
(ii) Applying the solution from step (i) to at least one side of the porous layer of the asymmetric SIS membrane; preferably, the asymmetric SIS membrane is immersed in the solution obtained in step (i);
(iii) Applying an exosome to at least one side of the porous layer of the asymmetric SIS membrane obtained in step (ii);
(iv) Drying the asymmetric SIS film obtained in step (iii) to obtain the composition.
(7) The production method according to (6), wherein the production step of the asymmetric SIS film comprises:
immersing a part of the SIS film into liquid nitrogen for cold quenching treatment, wherein a compact layer is formed on the part of the SIS film immersed into the liquid nitrogen, and a porous layer is formed on the part of the SIS film not immersed into the liquid nitrogen, so that the SIS film with an asymmetric structure is obtained;
preferably, the step of preparing the asymmetric SIS film further comprises: and (3) carrying out freezing treatment and freeze-drying treatment on the SIS film after the cold quenching treatment to obtain the asymmetric SIS film.
(8) The production method according to (6) or (7), wherein the exosome is an exosome derived from mesenchymal stem cells; preferably, the exosomes are exosomes derived from bone marrow mesenchymal stem cells.
(9) Use of the composition according to any one of (1) to (5) or the composition obtained by the production method according to any one of (6) to (8) in at least one of the following (a) to (c):
(a) As or to prepare osteogenic biological material;
(b) As or in the preparation of biological materials that promote tissue healing;
(c) As or in the preparation of biological materials for the treatment of bone defects.
(10) A method of treating a bone defect or promoting tissue healing or bone regeneration, wherein the method comprises the step of administering the composition according to any one of (1) - (5) or the composition obtained according to the method of preparation of any one of (6) - (8) to a subject.
ADVANTAGEOUS EFFECTS OF INVENTION
In some embodiments, the composition provided by the present disclosure provides, for the first time, an SIS membrane with an asymmetric structure, wherein the asymmetric SIS membrane covers a bone defect area, the porous structure of the porous layer of the SIS membrane can provide a suitable three-dimensional space for adhesion and proliferation of bone cells, and the dense layer of the asymmetric SIS membrane can effectively isolate soft tissues, avoid invasion of epithelial cells, fibroblasts and the like, and provide a stable growth space for proliferation and growth of the bone cells.
Compared with the existing SIS film loaded with exosomes, the porous layer of the asymmetric SIS film has high porosity, can remarkably improve the loading rate of exosomes on the SIS film, provides a proper microenvironment for healing and remodeling of bone tissues, and has remarkably improved capability of inducing bone regeneration.
In some specific embodiments, the porous layer and the dense layer of the asymmetric SIS membrane are loaded with exosomes, so that the loading amount of the exosomes on the asymmetric SIS membrane is further improved, and the exosomes loaded on one side of the porous layer can further promote tissue healing and growth of a soft tissue region.
In some embodiments, exosomes are supported on an asymmetric SIS membrane by a fusion polypeptide, and the fusion polypeptide in the present disclosure can achieve effective recruitment of exosomes, improve the binding rate and binding stability of exosomes on the asymmetric SIS membrane, enable exosomes to be released after being maintained on the asymmetric SIS membrane for a certain time, and effectively exert the effects of exosomes in promoting osteogenic differentiation and inducing bone regeneration.
In some embodiments, the fusion polypeptide of the present disclosure is a rigid linking peptide (PAPAP) linking exosome binding peptide CP05 (CRHSQMTVTSRL) to collagen binding peptide (DARKESVQK or CRHSQMTVTSRL). The rigid connecting peptide (PAPAAP) is rich in proline, can provide structural flexibility and maintain the original structure of each functional domain, maintain the fixed distance between the domains and keep the independent functions, and has positive effects on the combination of exosomes and asymmetric SIS membranes at two ends.
In some preferred embodiments, the exosomes in the present disclosure are derived from bone marrow mesenchymal stem cells, have an important role in inducing osteogenic differentiation and bone regeneration of BMSCs, can effectively activate bone tissue repair processes, promote survival, proliferation and osteogenic differentiation of cells, and have a positive effect on healing and reconstruction of bone defects.
In some embodiments, the present disclosure provides methods of preparing compositions that provide efficient, simple to operate, and easy to implement processing techniques for the preparation of SIS molds having asymmetric structures and modification of asymmetric SIS films.
In some embodiments, the present disclosure provides a method for preparing an asymmetric SIS membrane, by immersing an asymmetric SIS membrane portion in liquid nitrogen to perform a cold quenching treatment, a double-layer asymmetric structure formed by a dense layer and a porous layer can be obtained, a processing process for preparing a GBR membrane from the SIS membrane is realized for the first time, and the method has an important meaning for improving a clinical treatment effect of a GBR technology.
In some specific embodiments, the cold-quenched SIS film is further subjected to a freezing and lyophilization process to stabilize the structure of the asymmetric SIS film, resulting in an asymmetric SIS film with good tensile properties.
Drawings
FIG. 1 shows a schematic representation of SIS-P1P2-EXO membranes guiding bone regeneration, wherein the SIS-P1P2-EXO membrane is an exosome-loaded asymmetric SIS membrane mediated by fusion polypeptides.
Fig. 2 shows a 3D view of the molecular structure of fusion polypeptides P1 and P2, wherein the tertiary structure of fusion polypeptide P1 (Peptide 1) and fusion polypeptide P2 (Peptide 2) was predicted by the protein analysis software Robetta and visualized by VMD. P1: DARKESVQKPAPAPPAPAPCRHSQMTVTSRL; p2: LRELHLNNNPAPAPPAPAPCRHSQMTVTSRL.
Figure 3 shows the morphological characterization of an asymmetric SIS film, in figure 3 SEM images of a dense layer (×2000), a porous layer (×2000) and a cross section (×2000) in that order; scale bar: 20 μm.
Figure 4 shows a characterization of an asymmetric SIS membrane surface shown and modified. Wherein, (a) shows the exosome morphology observed by TEM. (B) The particle size distribution of the exosomes as measured by NanoSight analysis is shown. (C) Western blot analysis of exosome markers (CD 63, CD81, alix and CD 9) is shown. Cells were stained with cytochrome c. (D) SEM images of exosome-modified asymmetric SIS membranes (SIS-EXO membranes), fusion polypeptide-mediated exosome-modified asymmetric SIS membranes (SIS-P1P 2-EXO membranes) are shown. (E) The distribution of DiR-labeled exosomes on the surface of SIS-EXO membrane and SIS-P1P2-EXO membrane is shown. P1 was labeled with FITC and P2 was labeled with RhodaminB. Scale bar: 50 μm.
Figure 5 shows internalization of BMSCs to exosomes and in vitro biocompatibility of SIS membranes, SIS-EXO membranes and SIS-P1P2-EXO membranes. (A) BMSCs were incubated with DiI-labeled exosomes for 8 hours, 12 hours, 24 hours, 48 hours and 72 hours. Nuclei of BMSCs were stained with DAPI. (B) CCK-8 analysis of proliferation potency of BMSCs. * p <0.05; * P <0.01. In FIG. 5, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
FIG. 6 shows the effect of SIS-P1P2-EXO membranes, SIS membranes and the blank on osteogenic differentiation of BMSCs. (A) Protein expression levels of BMP2, OCN, ALP and OPN are shown by Western blotting. Protein levels were quantified by densitometry and normalized to GAPDH levels. (B) RNA expression of osteogenic related genes (BMP 2, OCN, ALP and OPN) is shown. * p <0.05; * P <0.01. In FIG. 6, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
FIG. 7 shows representative immunofluorescent staining of osteogenic markers (BMP 2, OCN, ALP, and OPN). The results of the BMP2, OCN, ALP, OPN, p-Akt, akt and nuclei (DAPI staining) are shown separately. In FIG. 7, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
Figure 8 shows that exosomes induce activation of PI3K/Akt signaling pathways. (A) Western blot of p-Akt and total Akt in BMSCs cultured for 5 days. GAPDH expression was used as a normalization control. (B) Immunofluorescent staining of p-Akt and total Akt in BMSCs cultured for 5 days. Wherein blank, SIS-EXO, SIS-P1P2-EXO indicates that no inhibitor is added during the culture; blank +LY, SIS-EXO +LY, SIS-P1P2-EXO +LY represents addition of inhibitor during culture. In FIG. 8, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
FIG. 9 shows a general assessment of in vivo performance after 12 weeks of SIS membrane, SIS-EXO membrane, biogide membrane and SIS-P1P2-EXO membrane implantation. (A) Representative three-dimensional (3D) reconstructions and sagittal images of critical dimension rat skull full-thickness defects are shown. (B) Bone volume fraction (BV/TV) of the defect area is shown. (C) Aggregate dates for Bone Mineral Density (BMD) of the defect area are shown. * p <0.05; * P <0.01. In FIG. 9, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
Fig. 10 shows histological and immunohistochemical analysis of newly formed bone at 12 weeks. (A) shows H & E staining of tissue sections. (B) Masson-Goldner staining of tissue sections is shown. (C) Immunohistochemical staining of the osteogenic markers COL1-A1 and OPN is shown. In FIG. 10, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
Fig. 11 shows photographs of SIS films before and after liquid nitrogen quenching.
FIG. 12 shows tensile strength of SIS films, SIS-EXO films, SIS-P1P2-EXO films, biogide films in dry and wet state. In FIG. 12, SIS is an asymmetric SIS membrane, SIS-EXO is an exosome-modified asymmetric SIS membrane, and SIS-P1P2-EXO is a fusion polypeptide-mediated exosome-modified asymmetric SIS membrane.
Figure 13 shows the binding capacity of recombinant peptides on SIS membrane surface observed by CLSM method. SIS membranes were immersed in four different concentrations of recombinant peptide solutions.
FIG. 14 shows the results of CLSM observations of fusion polypeptide and exosome release on SIS-P1P2-EXO membranes. Wherein, P1 is marked by FITC, P2 is marked by RhodaminB, and exosomes are marked by DiR; ruler: 50 μm.
Detailed Description
Definition of the definition
The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or specification may refer to "one" but may also refer to "one or more", "at least one" and "one or more".
As used in the claims and specification, the words "comprise," "have," "include" or "contain" mean including or open-ended, and do not exclude additional, unrecited elements or method steps.
Throughout this application, the term "about" means: one value includes the standard deviation of the error of the device or method used to determine the value.
Although the disclosure supports the definition of the term "or" as being inclusive of alternatives and "and/or", the term "or" in the claims means "and/or" unless expressly indicated otherwise as being exclusive of each other, as defined by the alternatives or alternatives.
As used in this disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, repetition, deletion, or addition of one or more amino acids or nucleotides. In the present disclosure, the term "mutation" refers to a change in nucleotide sequence or amino acid sequence. In some embodiments, a "mutation" of the present disclosure may be selected from "conservative mutations", "semi-conservative mutations", "non-conservative mutations". In the present disclosure, the term "non-conservative mutation" or "semi-conservative mutation" may be a mutation that causes loss or partial loss of function of a protein. The term "conservative mutation" refers to a mutation that normally maintains the function of a protein. Representative examples of conservative mutations are conservative substitutions.
As used in this disclosure, "conservative substitutions" typically exchange one amino acid at one or more sites of a protein. Such substitutions may be conservative. Specific examples of the substitution to be regarded as a conservative substitution include substitution of Ala to Ser or Thr, substitution of Arg to Gln, his or Lys, substitution of Asn to Glu, gln, lys, his or Asp, substitution of Asp to Asn, glu or Gln, substitution of Cys to Ser or Ala, substitution of Gln to Asn, glu, lys, his, asp or Arg, substitution of Glu to Gly, asn, gln, lys or Asp, substitution of Gly to Pro, substitution of His to Asn, lys, gln, arg or Tyr, substitution of Ile to Leu, met, val or Phe, substitution of Leu to Ile, met, val or Phe, substitution of Lys to Asn, glu, gln, his or Arg, substitution of Met to Ile, leu, val or Phe, substitution of Phe to Trp, tyr, met, ile or Leu, substitution of Ser to Thr or Ala, substitution of Thr to Ser or Ala, substitution of Trp to Phe or Tyr, substitution of Tyr to His, phe or Trp, and substitution of Val to Met, ile or Leu. In addition, conservative mutations include naturally occurring mutations resulting from individual differences, strains, species differences, and the like from which the gene is derived.
"sequence identity" and "percent identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or by deleting nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or by deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
Illustratively, in the present disclosure, two or more sequences or subsequences have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residue "sequence identity" or "percent identity" when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. The judgment/calculation of "sequence identity" or "percent identity" may be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (i.e., nucleic acids or polypeptides).
As used in this disclosure, the term "reverse complement" (Reverse Complementary Sequence) means: a sequence which is opposite to the sequence of the original polynucleotide and which is also complementary to the sequence of the original polynucleotide. Illustratively, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAGT.
As used in this disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments or may be an integral part of a larger nucleotide sequence structure, derived from nucleotide sequences that are separated at least once in number or concentration, and capable of identifying, manipulating and recovering sequences and their constituent nucleotide sequences by standard molecular biological methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or whole fragments), or may be a component or constituent of a larger nucleotide structure, such as an expression vector or polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. A "recombinant polynucleotide" or "recombinant nucleic acid molecule" belongs to one of the "polynucleotides".
As used in this disclosure, the term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide may be included in a suitable vector, and the vector may be used for transformation into a suitable host cell. The polynucleotide is then expressed in a recombinant host cell to produce, for example, "recombinant polypeptides," "recombinant proteins," "fusion proteins," and the like.
As used in this disclosure, the terms "linker" and "linker" may be used interchangeably and are capable of linking the same or different polypeptides or amino acids.
The connecting peptide includes flexible connecting peptide and rigid connecting peptide. In embodiments of the present disclosure, the linker peptide is selected from rigid linker peptides. Wherein, preferably, the rigid linking peptide is selected from: (Pro Ala Pro Ala Pro) n, n=an integer between 1 and 6. More preferably, the rigid linking peptide of the present disclosure is selected from (Pro Ala Pro Ala Pro) 2 。
As used in this disclosure, "exosomes (Extracellular Vesicles, EVs)" refer to vesicle-like bodies of bilayer membrane structure produced by cells via the paracrine pathway, varying in diameter from 40nm to 1000 nm. Exosomes are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk), carrying various proteins, lipids, etc. related to cell sources, and are involved in intercellular communication, cell migration, angiogenesis, immunomodulation, etc. In some specific embodiments, the exosomes are derived from mesenchymal stem cells, more preferably bone marrow mesenchymal stem cells (BMSCs).
As used in this disclosure, the term "high stringency conditions" refers to prehybridization and hybridization in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 65 ℃.
As used in this disclosure, the term "very high stringency conditions" refers to prehybridization and hybridization in 5X SSPE (saline sodium phosphate EDTA), 0.3% sds, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. Finally, the carrier material was washed three times, 15 minutes each, with 2 XSSC, 0.2% SDS at 70 ℃.
As used in this disclosure, the term "quench treatment" refers to a process of subjecting an SIS film to a phase inversion treatment using liquid nitrogen as a quench agent (sequencer). In the cold quenching process, liquid nitrogen is utilized to quickly form ice crystals from the moisture contained in the contact area, and abnormal expansion is utilized to further form a loose structure in the SIS film.
In the present disclosure, the terms "fusion polypeptide modified SIS membrane" and "fusion polypeptide mediated SIS membrane" are synonymous and may be used interchangeably unless specifically emphasized.
In this disclosure, the terms "loaded exosome", "modified exosome" and the like are intended to be used interchangeably unless specifically emphasized.
In this disclosure, the term "porous layer" may also be referred to as "Loose layer" unless specifically emphasized.
In the present disclosure, the exosome-loaded SIS-EXO films referred to in the examples, as well as fusion polypeptide-mediated exosome-loaded SIS-P1-EXO films, are made using asymmetric SIS films unless specifically emphasized.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Technical proposal
In the technical scheme of the disclosure, the meanings represented by the numbers of the nucleotide and amino acid sequence table in the specification are as follows:
SEQ ID NO:1 shows the amino acid sequence of exosome binding polypeptide CP05 (CRHSQMTVTSRL);
SEQ ID NO:2 shows the amino acid sequence of a type I collagen binding peptide (DARKESVQK);
SEQ ID NO:3 shows the amino acid sequence of a type iii collagen binding peptide (LRELHLNNN);
SEQ ID NO:4 shows the amino acid sequence of a connecting peptide (PAPAP);
SEQ ID NO:5 shows the amino acid sequence of fusion polypeptide P1 (DARKESVQKPAPAPPAPAPCRHSQMTVTSRL);
SEQ ID NO:6 shows the amino acid sequence of fusion polypeptide P2 (LRELHLNNNPAPAPPAPAPCRHSQMTVTSRL);
SEQ ID NO:7 shows the forward primer sequence for amplifying the BMP-2 gene;
SEQ ID NO:8 shows the reverse primer sequence for amplifying the BMP-2 gene;
SEQ ID NO:9 shows the forward primer sequence of the amplified PDGFA gene;
SEQ ID NO:10 shows the reverse primer sequence for amplifying the PDGFA gene;
SEQ ID NO:11 shows the forward primer sequence for amplifying the ALP gene;
SEQ ID NO:12 shows the reverse primer sequence for amplifying the ALP gene;
SEQ ID NO:13 shows the forward primer sequence for amplifying the OCN gene;
SEQ ID NO:14 shows the reverse primer sequence for amplifying the OCN gene;
SEQ ID NO:15 shows the forward primer sequence for amplifying the OPN gene;
SEQ ID NO:16 shows the reverse primer sequence for amplifying the OPN gene;
SEQ ID NO:17 shows the forward primer sequence for amplifying the COL1-A1 gene;
SEQ ID NO:18 shows the reverse primer sequence for amplifying the COL1-A1 gene;
SEQ ID NO:19 shows the forward primer sequence for amplifying PTEN gene;
SEQ ID NO:20 shows the reverse primer sequence of the amplified PTEN gene;
SEQ ID NO:21 shows the forward primer sequence for amplifying the gsk3β gene;
SEQ ID NO:22 shows the reverse primer sequence for amplifying the gsk3β gene;
SEQ ID NO:23 shows the forward primer sequence for amplifying the GAPDH gene;
SEQ ID NO:24 shows the reverse primer sequence for amplifying the GAPDH gene.
Table 1 sequences of fusion polypeptides shown in the present disclosure
TABLE 2 primer sequences employed in the present disclosure
Unless otherwise emphasized, the following general experimental methods (a) - (S) employed in the examples of the present disclosure were as follows:
(A) Synthesis of fusion polypeptides
Fusion polypeptides were synthesized commercially using Fmoc solid phase peptide synthesis (Shanghai Jier Biochemical Co., ltd., china). The sequence of the fusion polypeptide 1 is shown in SEQ ID NO:5, labeled with Fluorescein Isothiocyanate (FITC); the sequence of the fusion polypeptide 2 is shown in SEQ ID NO:6, which is labeled with Rhodamine B (RB). The peptides were purified to at least 90% purity and analyzed by mass spectrometry. The tertiary molecular structure of both fusion polypeptides was predicted using the protein analysis software Robetta.
(B) Preparation of asymmetric SIS film:
the asymmetric structure of SIS films is obtained by cold quenching in liquid nitrogen and subsequent lyophilization to produce a porous structure. Specifically:
and (3) immersing the SIS film part into liquid nitrogen for cold quenching treatment, wherein a compact layer is formed on the SIS film part immersed into the liquid nitrogen, and a porous layer is formed on the SIS film part not immersed into the liquid nitrogen, so that the SIS film with an asymmetric structure is obtained. Illustratively, the SIS film is contacted on one side directly with a liquid nitrogen portion, and because the SIS film has pores, liquid nitrogen permeates the SIS film to form an immersed portion, while the portion of the SIS film not contacted by liquid nitrogen forms an uninverted portion.
After a period of cold quenching (e.g., 20-40 seconds), the film is prepared as an asymmetric SIS film by taking out and freezing at-80℃ (e.g., 24-36 hours) followed by lyophilization in a lyophilizer.
(C) Cell culture and conditioned medium collection:
rat bone mesenchymal stem cells (BMSCs, 1×10 5 Cyagen Biosciences, china) was inoculated on SIS, SIS-EXO and SIS-P1P2-EXO membranes and cultured in proliferation medium (DMEM) containing Dulbecco's modified Eagle's, 10% (v/v) Fetal Bovine Serum (FBS), 100U/ml penicillin G and 100mg/ml streptomycin for 7 days. The medium was changed every 3 days.
(D) Isolation of exosomes:
exosomes were isolated from BMSCs by ultracentrifugation. The culture medium of bone marrow mesenchymal stem cells was collected in a conical tube for later use. Several centrifugation and filtration steps were performed to purify the exosomes. Briefly, the medium was centrifuged at 800g for 30 min to eliminate dead cells and at 12000g for 60 min, and then filtered through a 0.22 μm filter to remove cell debris. The supernatant was then ultracentrifuged at 100,000g for 90 minutes and washed with 100,000g of PBS for 90 minutes.
(E) Morphology observation of exosomes:
a transmission electron microscope (TEM; nova Nano TEM 430, netherlands) was used to observe the morphology of exosomes. Concentrated exosomes were fixed with 4% paraformaldehyde for 30 min. The mixture was then dropped onto a carbon coated copper mesh and dried for 15 minutes. The dried mixture was stained twice (5 minutes each) with 1% uranyl acetate. Images were obtained at 120kV using TEM. The particle size and distribution of the exosomes were measured by nanoparticle tracking analysis (NAT, nanoSight Ltd, malvern, UK). NTA analysis software (Nanoparticle Tracking Analysis, version 2.3) was used for analysis. Exosome specific markers such as CD63, CD81, CD9 and Alix were identified by western blotting.
(F) Exosome-modified asymmetric SIS membrane:
the asymmetric SIS membrane was cut into discs of 10 mm/35 mm diameter, matched to the pore size of a 24-well/6-well plate, and immersed overnight in a mixed suspension of fusion polypeptide solution and exosomes. Specifically:
after mixing and incubating the fusion polypeptide solution and the exosome suspension for 12 hours at 4 ℃, immersing the asymmetric SIS membrane into the mixed suspension for 24 hours, and then lyophilizing again for 6 hours to prepare the SIS-P1P2-EXO membrane.
Soaking the asymmetric SIS film in the suspension of exosomes to obtain the SIS-EXO film.
(G) Stability analysis of fusion polypeptide and exosomes:
to test for release of fusion polypeptides and exosomes, FITC-labeled and rhodamine B-labeled fusion polypeptides were coated on SIS membrane surfaces. Fusion polypeptide modified SIS membranes were immersed in exosome suspensions overnight at 4 ℃ prior to use. Samples were observed using a confocal laser scanning microscope (CLSM, nikon Air Confocal, australia).
(H) Exosome uptake assay:
exosomes were labeled with DiI (Sigma-Aldrich). Mu.l of exosome suspension was mixed with 5. Mu.l of DiI for 1 hour. The labeled exosomes were then washed in PBS and centrifuged at 100,000g for 1 hour. The exosomes were then incubated with BMSCs for 8 hours, 12 hours, 24 hours, 48 hours and 72 hours, respectively. After incubation, cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min and washed again. DAPI solution was used to stain nuclei. Images were taken with CLSM (Nikon Air Confocal, australia).
(I) Determination of mechanical properties:
SIS, SIS-EXO, SIS-P1P2-EXO and Biogide films were cut into suitable samples (15 mm wide by 20 mm long) for measuring tensile strength. Measurements were made using a limiting tensile tester (3367, instron, usa) under wet (pre-soaked in PBS for 0.5 hours) and dry conditions at a crosshead speed of 10 mm/min. The tensile strength of the test specimens was then calculated and recorded based on the Blue Hill system.
(J) Morphological observations of fusion polypeptides and exosome-modified asymmetric SIS films
The surface morphology of SIS and SIS-P1P1-EXO films was observed by scanning electron microscopy (SEM; novaNanoSEM430, FEI, netherlands). The samples were gold sputter coated in an argon atmosphere using a sputter coater (K575 XD, emitch, england) prior to scanning. Confocal laser scanning microscopy (CLSM; FV1000, olympus, japan) was used to observe target binding of fusion polypeptides and exosomes on SIS membrane surfaces.
(K) Cell proliferation assay:
cell viability was measured by the CCK-8 assay. SIS, SIS-EXO and SIS-P1P2-EXO samples were cut into 6mm diameter discs and placed in 96-well plates. Subsequently, BMSC cells were seeded in 96-well plates (2000 cells/well). Three replicates were performed for each group. 1. After 3, 5, 7 days, 10. Mu.l of CCK-8 reagent (Solarbio, china) was added to each well and incubated for 4h at 37 ℃. OD values were calculated at 450nm absorbance.
(L) quantitative real-time polymerase chain reaction (qRT-PCR) analysis:
BMSCs were cultured in osteogenic medium for 7 days. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and then GoScript was used TM Reverse Transcription Mix (Promega, USA) was reverse transcribed. qRT-PCRKit (Qiagen, germany) examined the mRNA expression of bone morphogenic protein 2 (BMP-2), osteocalcin (OCN), alkaline phosphatase (ALP) and Osteopontin (OPN). GAPDH was used as a reference. Fold differences were calculated using the ΔΔct method. The primers are listed in Table 1.
(M) Western blot analysis:
will be 1X 10 5 After three days of BMSCs inoculation into six well plates with sterilized SIS membrane, SIS-EXO membrane and SIS-P1P2-EXO membrane, the medium in the wells was replaced with osteoinductive medium. Cell lysates were diluted with protein loading buffer (4X) and heated at 95℃for 10 minAnd (3) a clock. The protein extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Sigma, usa) at 300mA for 90 minutes. Membranes were blocked with 5% bsa (Sigma, usa) for 1 hour at room temperature and incubated with primary antibody (Abcam, uk) overnight at 4 ℃. The membrane was then incubated with secondary antibody (Abcam, uk) for 70 minutes at 37 ℃. One antibody includes anti-BMP 2 (1:1000, abcam), anti-OCN (1:1000, abcam), anti-ALP (1:1000, abcam), anti-OPN (1:1000, abcam), anti-p-Akt (1:1000, abcam), and anti-Akt (1:1000, abcam). All values were normalized by GAPDH (1:1000, abcam).
(N) immunofluorescent staining:
for immunofluorescence analysis, cells were fixed with 4% formaldehyde for 30 min at room temperature. After 10 minutes of permeabilization of the cells with 0.25% Triton X-100 (Sigma, USA), the corresponding antibodies were then used to immunostain BMP-2, OCN, ALP, OPN, p-Akt, and Akt (Abcam, UK). The cells were further labeled with a secondary antibody coupled to fluorescein isothiocyanate. DAPI is used to stain nuclei. Cells were then imaged with CLSM (nikonai confocal, australia).
(O) PI3K/Akt signal inhibition:
LY294002 is a highly selective PI3K inhibitor, purchased from Sigma-Aldrich (St. Louis, mitsui, U.S.A.), and dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 100mM according to the protocol. To confirm that PI3K/Akt signals are involved in exosome-mediated BMSC effects, cells were placed on the blank, SIS-EXO and SIS-P1P2-EXO group samples with or without LY294002 for 5 days; wherein blank, SIS-EXO, SIS-P1P2-EXO indicates that no inhibitor is added during the culture; blank +LY, SIS-EXO +LY, SIS-P1P2-EXO +LY represents addition of inhibitor during culture. Western blot and immunofluorescent staining assays were then performed as described above.
(P) animals and surgery:
all animal experiments were approved by the animal ethics and welfare Committee of Tianjin medical university (SYXK: 2019-0004). Male 6 week old SD rats were randomly divided into a blank group, SIS membrane group, SIS-EXO membrane group, bio-Gide membrane group and SIS-P1P2-EXO membrane group 5 group. A critical dimension defect with a diameter of 8mm was made in the skull with a diamond needle (Hager Meisinger co., ltd, germany) at slow drilling. The membrane is then implanted into the defect and the incision is sutured.
(Q) micro CT scan evaluation:
each group of rats was sacrificed by isoflurane inhalation 12 weeks after surgery. Bone regeneration in the area of the skull defect was assessed by microscopic CT analysis (SkyScan 1276, germany). After three-dimensional (3D) visualization, the bone volume/total volume ratio (BV/TV) and Bone Mineral Density (BMD) were checked using CTAn software.
(R) histological and immunohistochemical analysis:
after decalcification for 30 days, the tissues were embedded in paraffin and sectioned (thickness 5 μm). Histological analysis was performed by hematoxylin eosin (H & E) and Masson-Goldner (Solarbio, china) staining. For immunohistochemistry, a primary antibody against COL1-A1 and against OPN (1:200 dilution; abcam, UK) was used. Immunoreactivity was detected using fluorescence conjugated goat anti-rabbit IgG (1:200 dilution; solarbio, beijing, china). Images were observed using a digital slice scanning system (NanoZoomer, hamamatsu, japan).
(S) statistical analysis:
all results are expressed as mean ± standard deviation of each group. Analysis was performed by one-way analysis of variance and Tukey post-hoc test. For all tests, if p <0.05 (x), the difference was considered significant.
Examples
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.
Example 1: design and Structure prediction of fusion polypeptides
The predicted pseudo 3D structure of the fusion polypeptide is shown in fig. 2. The two fusion polypeptides consist of three parts, CP05 (CRHSQMTVTSRL), collagen binding peptide (type I collagen: DARKESVQK or type III collagen: LRELHLNNN) and connecting peptide (PAAP). The prediction map shows that the spatial structure of the two fusion polypeptides is different (fig. 2). The backbone structure of fusion polypeptide P1 is relatively compact because the alpha-helical structure is rich in hydrogen bonds. Fusion polypeptide P2 has a random coil structure and better mobility, which allows CP05 to oscillate over a wider range.
In the present disclosure, pro-rich (proline rich) linker peptide (PAPAAP) was chosen as the linker peptide for constructing the fusion polypeptide. The linker peptide is an important factor in constructing fusion polypeptides, providing structural flexibility and preserving the original structure of the functional domain. Pro-rich linker peptide (PAAP) is one of the rigid linkers peptides that has been successfully used to maintain a fixed distance between domains and to maintain their independent function, which is useful for the construction of fusion polypeptides. The non-helical rigid linker peptide (PAPAP) exhibits sufficient rigidity and helps to reduce inter-domain interference due to Pro-rich sequences, which imposes strong conformational constraints and increases the rigidity of the linker peptide. Furthermore, to achieve proper separation of the functional domains and avoid interference with each other, the sequence of the connecting peptide is repeated twice, helping the fusion polypeptide to recruit and anchor the exosomes to the SIS membrane surface.
Example 2: morphology of asymmetric SIS films
The asymmetric structure of the asymmetric SIS film is formed by liquid nitrogen quenching. This process can result in a temperature gradient between the upper and lower layers of the SIS film. It has been shown that the number of nuclei initially formed at higher temperatures is lower than the number of nuclei formed at lower freezing temperatures. Thus, the upper layer of SIS film not contacted with liquid nitrogen forms fewer ice crystal nuclei, while the lower layer contacted with liquid nitrogen forms more ice crystal nuclei. This difference in the number of ice nuclei resulted in larger pores in the upper layer (porous layer) and smaller pores in the lower layer (dense layer) after lyophilization (fig. 3).
The surface morphology of the asymmetric SIS film was characterized by SEM images. As shown in fig. 3, the cross-section of the asymmetric SIS membrane exhibits an asymmetric structure, including a dense layer and a porous layer; the dense layer shows a dense and flat surface morphology. The porous layer has macropores communicating with each other. A photograph of the asymmetric SIS film after liquid nitrogen quenching treatment is shown in fig. 11. These results indicate that asymmetric SIS films were successfully prepared.
Adequate mechanical properties are critical to maintaining bone formation space. The tensile strength of the asymmetric SIS film when dry and wet is shown in figure 12. The tensile strength of asymmetric SIS, SIS-EXO and SIS-P1P2-EXO membrane sets was similar to that of Biogide. There was no significant difference between the groups. The tensile strength of all groups decreased slightly under wet conditions.
Example 3: characterization of exosome-modified asymmetric SIS membrane surfaces
Exosomes were extracted from the culture medium of BMSCs by ultracentrifugation. TEM, NTA analysis and western blot were used to characterize exosomes derived from BMSCs. The results indicate that most of these particles exhibit a cup-shaped or round morphology (a and B in fig. 4) ranging in size from 50 to 150nm, indicating the presence of exosomes. Western blot analysis (FIG. 4C) showed that the exosome characteristic surface markers CD63, CD9, CD81 and Alix were positive and that cytochrome C (cell marker) was negative. These results indicate that exosomes were successfully isolated.
The exosome particles distributed on the surface of SIS-EXO and SIS-P1P2-EXO membranes exhibited cup-shaped morphology of varying sizes (FIG. 4D). To further demonstrate whether exosomes and fusion polypeptides were modified on asymmetric SIS membranes, a set of CLSM images validated the loading of exosomes and fusion polypeptides (fig. 4E). The CLSM image showed that DiR-labeled exosomes were evenly distributed on SIS-P1P2-EXO membrane, on which only dispersed DiR-labeled exosomes were found (fig. 4E). More DIR-tagged exosomes on SIS-P1P2-EXO membranes than on SIS-EXO membranes, indicating efficient recruitment of fusion polypeptides.
To further elucidate the release of fusion polypeptides and exosomes, CLSM observed changes in fusion polypeptides and exosomes on SIS-P1P2-EXO membranes (fig. 14). On the first day, a large number of fusion polypeptides and exosomes were observed on SIS-P1P2-EXO membranes. Over time, fusion polypeptides and exosomes bound to the SIS-P1P2-EXO membrane surface decrease. After 10 days, the levels of fusion polypeptide and exosomes were significantly reduced, but were still observed on SIS-P1P2-EXO membrane, indicating that fusion polypeptide and exosomes could be maintained for a certain period of time and released continuously.
These results indicate that exosomes bind efficiently to the fusion polypeptide modified asymmetric SIS membrane. The connecting peptide (PAPAP) plays an important role in the modification of SIS membranes by fusion polypeptides, which acts as a passive connecting peptide to maintain the distance between the collagen binding peptide and CP05 and to provide greater structural stability to the fusion polypeptide.
To elucidate the effect of the concentration of the fusion polypeptide on the binding of the fusion polypeptide to the asymmetric SIS membrane, the asymmetric SIS membrane was immersed in solutions of fusion polypeptides P1, P2 of different concentrations for concentration screening. The CLSM image (fig. 13) is displayed at 200×10 -6 Under the condition of the concentration of M, the fusion polypeptide P1 and the fusion polypeptide P2 can obtain good binding effect with an asymmetric SIS film.
Example 4: effects of bone marrow mesenchymal Stem cells on exosomes internalization and exosomes on cell proliferation
Exosomes can regulate the tissue repair process by stimulating cell proliferation and differentiation. To demonstrate that exosomes can be taken up by cells and have an effect on cell behaviour, BMSCs were incubated with DiI-labeled exosomes for 8 hours, 12 hours, 24 hours, 48 hours and 72 hours, respectively. Fluorescence microscopy images showed that exosomes were gradually internalized by BMSC from 8 hours to 24 hours (fig. 5A). After 48 hours of incubation, a large number of exosomes were internalized and distributed in the perinuclear region (fig. 5A). The fluorescence intensity peaks at 72h, indicating that exosomes can be transported to the recipient cells in the early stages of tissue repair to modulate cellular behavior.
The OD values of the exosome treated groups were significantly higher than those of the blank and SIS groups (p < 0.05) as determined by CCK-8 to assess cell proliferation over a period of 7 days (FIG. 5B). The proliferation capacity of BMSCs was higher in the SIS-P1P2-EXO group than in the SIS-EXO group (P < 0.05), and there was no statistical difference between the blank group and the SIS group (P > 0.05).
The results show that the fusion polypeptide can enhance the retention and stability of exosomes and enhance the therapeutic effect. The SIS membrane modified by the fusion polypeptide and the exosome has ideal biocompatibility and stronger capability of promoting cell activity, which shows that the fusion polypeptide has potential application value in the aspect of biological material modification.
Example 5: promotion of osteogenic differentiation of BMSCs with fusion polypeptides and exosome-modified asymmetric SIS membranes
Osteogenic differentiation of BMSCs is regulated by factors such as BMP2, OCN, ALP, and OPN. In the present disclosure, western blot analysis showed that the expression levels of BMP2, OCN, ALP and OPN in BMSCs of SIS-P1P2-EXO and SIS-EXO were significantly up-regulated (P < 0.05) compared to SIS and the blank (FIG. 6A). Immunofluorescence further confirmed the up-regulation of osteogenic related marker expression at the protein level (fig. 7). IF staining showed a significant increase in fluorescence intensity of BMP2, OCN, ALP and OPN in the SIS-P1P2-EXO and SIS-EXO groups. Of the groups, the expression level of the osteogenic related protein marker of SIS-P1P2-EXO group was highest. The relevant expression levels of mRNA were confirmed by qRT-PCR analysis (fig. 6B). Consistently, qRT-PCR analysis showed that the SIS-P1P2-EXO group showed the highest levels of BMP2, OCN, ALP, and OPNmRNA (P < 0.05). In conclusion, SIS-P1P2-EXO membranes significantly promoted osteogenic differentiation of BMSCs.
Cell-material interactions and cell-cell communication involving the secretion of signal factors by various cells are the primary biological processes in bone healing and remodeling processes. The design of GBR membranes capable of providing an osteogenic microenvironment to induce osteogenic differentiation of BMSCs is of great importance for bone tissue regeneration. In the present disclosure, exosomes released early from SIS-P1P2-EXO membranes help to modulate intercellular communication and ultimately activate a range of cellular responses. SIS-P1P2-EXO membranes exhibited a higher osteogenic capacity than SIS-EXO membranes, indicating that the fusion polypeptides have a positive effect on exosome-mediated osteogenic differentiation of BMSCs.
Example 6: activating PI3K/Akt signaling pathway
This example further illustrates the molecular mechanism of exosomes to osteogenic differentiation of BMSCs. Studies have shown that exosomes secreted by human bone marrow mesenchymal stem cells can activate and increase osteogenic differentiation of a variety of signaling pathways (including Akt, erk1/2, and STAT 3) targeting mesenchymal stem cells. To observe the effect of exosomes on the PI3K/Akt pathway, we examined Akt and p-Akt levels using WB and IF assays (a and B in fig. 8). The P-Akt levels were higher in the SIS-P1P2-EXO and SIS-EXO groups than in the SIS and blank groups. The SIS-P1P2-EXO group showed the highest levels of P-Akt, indicating that the engineered recombinant peptide captured a large number of exosomes. However, up-regulation of p-Akt in BMSCs by exosomes was inhibited by culture of BMSCs with PI3K inhibitor (LY 294002). These results indicate that PI3K/Akt signaling pathways in BMSCs are activated by exosomes. Exosome-induced osteogenic differentiation of BMSCs is due, at least in part, to activation of PI3K/Akt signaling pathways.
Example 7: evaluation of in vivo bone regeneration ability of SIS-P1P2-EXO Membrane
To investigate the therapeutic potential of SIS-P1P2-EXO membranes for bone defect repair, critical dimension defects with a diameter of 8mm were created in animal experiments and the membranes were implanted into the defect area. As shown in fig. 9a, the micro CT images of all groups showed that new bone formation progressed from the edge of the defect to the center. When covered with SIS-P1P2-EXO film, the defect was filled with uniform mature bone (FIG. 9A). In particular, the surface morphology of the healing defect is very similar to that of the surrounding normal bone. In the control and SIS groups, only a small number of high density spots were observed. In addition, 3D reconstruction was performed by analyzing bone volume fraction (BV/TV) and Bone Mineral Density (BMD) (B and C in fig. 9). Quantification of CT images provided further evidence that new bone formation was significantly greater in SIS-P1P2-EXO groups than in the other four groups (P < 0.05). Representative microct images and quantitative analysis showed that SIS-P1P2-EXO membranes activated bone tissue repair processes effectively.
Example 9: SIS-P1P2-EXO membrane in vivo bone regenerationHistological assessment of competence
Histological analysis was performed to assess collagen and new tissue growth and lymphocyte infiltration. HE staining results showed that no apparent inflammatory tissue was seen in either group. The empty group defect areas were filled with large fibrous connective tissue, and no obvious signs of new bone formation were seen (a in fig. 10). In the SIS group, a small amount of newly formed bone tissue was observed in the center of the defect area. In the SIS-EXO and Biogide groups, the new trabecular bone was evenly distributed without invasion of fibrous connective tissue. SIS-P1P2-EXO group showed the best structural integrity among all groups. Collagen content is closely related to bone formation. Masson-Goldner staining showed that mature collagen and osteoid tissue formation was greater in the SIS-P1P2-EXO membrane group than in the other groups (FIG. 10B). Early-formed bone is reconstituted into regular lamellar bone, and substantial collagen matrix deposition occurs. IHC staining showed that SIS-EXO group formed more new bone than SIS and control group (fig. 10C). Higher expression of OPN and COL1-A1 was detected in the Biogide group. The highest expression level was found in SIS-P1P2-EXO group, which suggests that SIS-P1P2-EXO membrane exerts the strongest effect on bone regeneration.
The results show that the SIS-P1P2-EXO film can effectively prevent the fibrous connective tissue from growing to the defect area, and provides space for bone regeneration. Fusion polypeptides constructed in accordance with the present disclosure will prolong the duration of action of the exosomes and increase their stability. SIS-P1P2-EXO membranes exhibit the strongest bone repair because of the large number of exosomes captured by the fusion polypeptide. The excellent bone regeneration capability of the SIS-P1P2-EXO film is beneficial to solving the problems of bone deficiency, bone loss and insufficient alveolar bone quantity caused by trauma or inflammation in a dental implant area. In addition, the exosome fusion polypeptide provides a new therapeutic concept for decellularized tissue regeneration.
In this disclosure a method of preparing an asymmetric SIS film using a cold quenching process of liquid nitrogen was developed. Through modification of the fusion polypeptide, SIS membranes can specifically bind to BMSC-exosomes. These peptides promote the positive effect of exosomes on BMSC osteogenic differentiation. SIS-P1P2-EXO membranes are effective in reconstructing bone tissue defects and effecting cell-free bone regeneration within 12 weeks. Furthermore, the PI3K/Akt signaling pathway plays a key role in the osteogenic effects of SIS-P1P2-EXO membrane on BMSC. These results indicate that SIS-P1P2-EXO membranes have potential for clinical application as novel GBR membranes.
The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure and are not limiting of the embodiments of the present disclosure. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the claims of the present disclosure.
SEQUENCE LISTING
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Claims (10)
1. A composition, wherein the composition comprises a fusion polypeptide modified asymmetric SIS membrane and exosomes; the asymmetric SIS membrane has oppositely facing porous and dense layers with at least a portion of the exosomes being supported inside and/or on the surface of the porous layer.
2. The composition of claim 1, wherein the sequence of the fusion polypeptide comprises at least one of the group consisting of the sequences set forth in seq id no:
(i) Consists of the sequence as shown in SEQ ID NO:5 and the sequence shown as SEQ ID NO:6, a group consisting of the sequences shown in fig. 6;
(ii) Sequences in which there is one, two, three, four or five conservative substitutions compared to the sequences shown in (i);
preferably, the sequence of the fusion polypeptide consists of at least one of the group consisting of:
(i) Consists of the sequence as shown in SEQ ID NO:5 and the sequence shown as SEQ ID NO:6, a group consisting of the sequences shown in fig. 6;
(ii) There is one, two, three, four or five conservatively substituted sequences compared to the sequence set forth in (i).
3. A composition according to claim 1 or 2, wherein both ends of the fusion polypeptide bind the exosomes and the asymmetric SIS membrane, respectively;
preferably, the exosomes are derived from mesenchymal stem cells; more preferably, the exosomes are derived from exosomes of bone marrow mesenchymal stem cells.
4. A composition according to any one of claims 1 to 3, wherein the asymmetric SIS film is obtained by partial immersion of a SIS film in liquid nitrogen, the portion of the SIS film immersed in liquid nitrogen forming the dense layer and the portion of the SIS film not immersed in liquid nitrogen forming the porous layer;
alternatively, the asymmetric SIS membrane is obtained by partial immersion of the SIS membrane in dry form in liquid nitrogen;
preferably, the dense and porous layers of the asymmetric SIS membrane are each loaded with exosomes.
5. A composition according to any one of claims 1 to 4 wherein the composition is obtained by incubating the asymmetric SIS membrane with the fusion polypeptide and the exosomes.
6. A method of preparing the composition of any one of claims 1-5, wherein the method of preparation comprises (a) - (d) or (i) - (iv) as follows:
(a) Dissolving the fusion polypeptide into a solvent to obtain a solution containing the fusion polypeptide;
(b) Mixing and incubating the solution obtained in the step (a) and the exosome to obtain an incubation solution;
(c) Applying the incubation solution from step (b) to at least one side of the porous layer of the asymmetric SIS membrane; preferably, the asymmetric SIS membrane is immersed in the incubation solution obtained in step (b);
(d) Drying the asymmetric SIS film with the incubation solution on the surface to obtain the composition; or alternatively
(i) Dissolving the fusion polypeptide into a solvent to obtain a solution containing the fusion polypeptide;
(ii) Applying the solution from step (i) to at least one side of the porous layer of the asymmetric SIS membrane; preferably, the asymmetric SIS membrane is immersed in the solution obtained in step (i);
(iii) Applying an exosome to at least one side of the porous layer of the asymmetric SIS membrane obtained in step (ii);
(iv) Drying the asymmetric SIS film obtained in step (iii) to obtain the composition.
7. The method of manufacturing of claim 6, wherein the step of manufacturing the asymmetric SIS film comprises:
immersing a part of the SIS film into liquid nitrogen for cold quenching treatment, wherein a compact layer is formed on the part of the SIS film immersed into the liquid nitrogen, and a porous layer is formed on the part of the SIS film not immersed into the liquid nitrogen, so that the SIS film with an asymmetric structure is obtained;
preferably, the step of preparing the asymmetric SIS film further comprises: and (3) carrying out freezing treatment and freeze-drying treatment on the SIS film after the cold quenching treatment to obtain the asymmetric SIS film.
8. The preparation method according to claim 6 or 7, wherein the exosome is an exosome derived from mesenchymal stem cells; preferably, the exosomes are exosomes derived from bone marrow mesenchymal stem cells.
9. Use of a composition according to any one of claims 1 to 5 or a composition obtainable by a process according to any one of claims 6 to 8 in at least one of the following (a) - (c):
(a) As or to prepare osteogenic biological material;
(b) As or in the preparation of biological materials that promote tissue healing;
(c) As or in the preparation of biological materials for the treatment of bone defects.
10. A method of treating a bone defect or promoting tissue healing or bone regeneration, wherein the method comprises the step of administering to a subject a composition according to any one of claims 1-5 or a composition obtained according to the method of preparation of any one of claims 6-8.
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| CN102229646A (en) * | 2011-06-09 | 2011-11-02 | 北京大学第三医院 | Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application |
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| CN102229646A (en) * | 2011-06-09 | 2011-11-02 | 北京大学第三医院 | Amino acid sequence of bone marrow mesenchymal stem cell affinity peptide, screening method and application |
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