CN117919451A - Application of biological material related to desmoglein 2 and medicine for inhibiting pathological hyperfunction of bone regeneration - Google Patents
Application of biological material related to desmoglein 2 and medicine for inhibiting pathological hyperfunction of bone regeneration Download PDFInfo
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- CN117919451A CN117919451A CN202410323999.7A CN202410323999A CN117919451A CN 117919451 A CN117919451 A CN 117919451A CN 202410323999 A CN202410323999 A CN 202410323999A CN 117919451 A CN117919451 A CN 117919451A
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
- Epidemiology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immunology (AREA)
Abstract
The invention discloses application of biological materials related to desmoglein 2 in the field of genetic engineering and a medicament for inhibiting pathological hyperfunction of bone regeneration. The technical problem to be solved by the invention is how to provide the application of the biological material related to desmoglein 2 in inhibiting pathological hyperfunction of bone regeneration activity. In order to solve the technical problem, the invention provides application of biological materials related to desmoglein 2 and a medicament for inhibiting pathological hyperfunction of bone regeneration, wherein the biological materials related to desmoglein 2 can be used for preparing the medicament for inhibiting the pathological hyperfunction of bone regeneration, and the medicament consists of medicinal auxiliary materials and substances for down regulating encoding genes of desmoglein 2. The medicine provided by the invention can inhibit local cell osteogenic differentiation, reduce the number of osteoblasts, and effectively relieve the pathological damage of bone cartilage diseases with pathological hyperfunction of bone regeneration activity.
Description
Technical Field
The invention relates to application of biological materials related to desmoglein 2 in the field of genetic engineering and a medicament for inhibiting pathological hyperfunction of bone regeneration.
Background
Bone regeneration refers to the activity of skeletal tissue in physiological and pathological states to create new bone tissue, participate in skeletal system development, maintain skeletal system homeostasis, combat skeletal system aging, and promote repair of skeletal system injury. However, overactivity of bone regeneration may cause diseases such as osteoarthritis, osteophyte formation, and bone sclerosis. Therefore, the mechanism of bone regeneration is deeply understood, a target point of bone regeneration is sought, and fine regulation of the bone regeneration process is helpful for providing a new therapeutic strategy and method for the pathological hyperfunction related diseases of bone regeneration.
Disclosure of Invention
The technical problem to be solved by the invention is how to provide the application of the biological material related to desmoglein 2 in inhibiting pathological hyperfunction of bone regeneration activity. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides application of a biological material related to desmoglein 2 in preparing a medicament for inhibiting pathological hyperactive bone regeneration, wherein the biological material is any one of the following materials:
b1 An RNA molecule that inhibits or reduces or down-regulates expression of a gene encoding desmoglein 2 or a chemical modification of said RNA molecule;
B2 A gene encoding the RNA molecule of B1);
B3 An expression cassette comprising the gene of B2);
b4 A recombinant vector comprising the gene of B2), or a recombinant vector comprising the expression cassette of B3);
b5 A recombinant microorganism comprising the gene of B2), a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4);
B6 A transgenic animal tissue comprising B1) said RNA molecule, or a transgenic animal tissue comprising B2) said expression cassette;
b7 A transgenic animal organ containing the RNA molecule of B1), or a transgenic animal organ containing the expression cassette of B2).
The desmosomal glycoprotein 2 is any one of the following:
a1 A protein having an amino acid sequence of SEQ ID No. 1;
A2 A protein which is obtained by substituting and/or deleting and/or adding the amino acid residues in the amino acid sequence shown in the A1), has more than 80% of identity with the protein shown in the protein and has the same function;
a3 A fusion protein having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of the amino acid shown in either A1) or A2).
Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
The nucleotide sequences encoding the above proteins of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the above protein isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above protein and function as the above protein.
Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
In the above application B1) the RNA molecule targets mRNA transcribed from the gene encoding desmoglein 2.
In the application, the coding gene of the desmoglein 2 is SEQ ID No.2.
The invention also provides a medicine for inhibiting pathological hyperfunction of bone regeneration, which consists of pharmaceutical auxiliary materials and substances for down-regulating encoding genes of desmoglein 2.
In the above medicine, the substance is any one of the following:
b1 An RNA molecule that inhibits or reduces or down-regulates expression of the coding gene or a chemical modification of the RNA molecule;
B2 A gene encoding the RNA molecule of B1);
B3 An expression cassette comprising the gene of B2);
b4 A recombinant vector comprising the gene of B2), or a recombinant vector comprising the expression cassette of B3);
B5 A recombinant microorganism comprising the gene of B2), a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4).
The chemical modifier is a substance obtained by chemically modifying the RNA molecule. The chemical modification may include one or a combination of several selected from ribose modification, base modification, and phosphate backbone modification.
Among the above, the expression cassette containing a nucleic acid molecule as described in B3) refers to a DNA capable of expressing the above-described protein in a host cell. The expression cassette may also include single or double stranded nucleic acid molecules of all regulatory sequences necessary for expression of the nucleic acid molecules of any of the proteins described above. The regulatory sequences are capable of directing the expression of any of the above proteins in a suitable host cell under conditions compatible with the regulatory sequences. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include promoters and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector in order to ligate the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein, a ligated regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e.a nucleic acid sequence which is recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein. The promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The control sequence may also be a suitable transcription termination sequence, a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequences may also be suitable leader sequences, i.e., untranslated regions of mRNA which are important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the host cell of choice may be used in the present invention. The regulatory sequence may also be a signal peptide coding region which codes for an amino acid sequence attached to the amino terminus of the protein and which directs the encoded protein into the cell's secretory pathway. Signal peptide coding regions that direct the expressed protein into the secretory pathway of host cells used may be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein according to the growth of the host cell. Examples of regulatory sequences are those systems which are capable of opening or closing gene expression in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those which enable the amplification of a gene.
In the above application, the recombinant microorganism may be a recombinant adeno-associated virus expressing the shRNA or siRNA.
In the above application, the cell may be a mammalian cell.
The medicament is in the form of injection. The pharmaceutical excipients comprise physiologically or pharmaceutically acceptable carriers or excipients. Herein, "physiologically or pharmaceutically acceptable carrier" refers to those carriers and diluents which have no significant irritating effect on the organism and which do not impair the biological activity and performance of the agent in the pharmaceutical composition to be administered. The carrier materials herein include, but are not limited to, water soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (e.g., ethylcellulose, cholesterol stearate, etc.). Among them, preferred is a water-soluble carrier material. For preparing unit dosage forms into injectable preparations such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art, for example, water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. may be used. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added.
In the above medicament, the RNA molecule targets mRNA transcribed from the gene encoding desmoglein 2.
In the medicine, the coding gene of desmoglein 2 is SEQ ID No.2.
In the above medicament, the RNA molecule is siRNA, and the siRNA is SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or/and SEQ ID No.6.
In the above-mentioned medicament, the pathological hyperfunction of bone regeneration includes pathological hyperfunction of bone regeneration caused by one or more diseases of osteoarthritis, ectopic ossification and spinal fusion.
Experiments prove that DSG2 has correlation with the bone regeneration capability, viruses prepared by constructing adeno-associated virus vectors can inhibit local cell osteogenic differentiation, reduce the number of osteoblasts, and effectively relieve pathological damage of bone cartilage diseases with pathological hyperfunction of bone regeneration (such as osteoarthritis, ectopic ossification, spinal fusion and the like). Therefore, the compound can be used as an osteogenesis inhibitor for treating diseases such as osteoarthritis, ectopic ossification, spinal fusion and the like.
Drawings
FIG. 1 is a diagram UMAP showing the cell types of osteochondral stem cell populations in tissues with abnormal elevated light and heavy bone regeneration detected by single cell sequencing. Wherein red represents the osteochondral stem cell 1 population, yellow represents the osteochondral stem cell 2 population, green represents the osteochondral stem cell 3 population, cyan represents the osteochondral stem cell 4 population, blue represents the osteochondral stem cell 5 population, and pink represents the osteochondral stem cell 6 population. UMAP _1 represents the unified manifold approximation and projection dimension 1, and UMAP_2 represents the unified manifold approximation and projection dimension 2.
FIG. 2 shows the expression of a gene related to hardening in a tissue with abnormal hyperstimulation of bone regeneration. Wherein 1 is a tissue with abnormal hyperfunction of light bone regeneration, and 2 is a tissue with abnormal hyperfunction of heavy bone regeneration; POSTN is shown in the 1 st left-right picture, namely periostin, COL1A1 is shown in the 2 nd left-right picture, namely type I collagen alpha 1 chain, COL3A1 is shown in the 3 rd left-right picture, namely type III collagen alpha 1 chain, and SPP1 is shown in the 4 th left-right picture, namely secreted phosphoprotein 1.
FIG. 3 shows a population of osteochondral tissue stem cells highly expressing DSG2 with increased specificity during abnormal hyperregeneration of bone. Wherein the left graph shows the result of the bone cartilage tissue with the light abnormal hyperregeneration of bone, and the right graph shows the result of the bone cartilage tissue with the heavy abnormal hyperregeneration of bone. Purple indicates the transcriptional expression level of DSG 2. UMAP _1 of the left graph represents the unified manifold approximation and projection dimension 1, and UMAP_2 represents the unified manifold approximation and projection dimension 2. UMAP _1 of the right graph represents the unified manifold approximation and projection dimension 1, and UMAP_2 represents the unified manifold approximation and projection dimension 2.
FIG. 4 is a view of violin showing the transcriptional expression level of DSG2 in osteochondral tissue with abnormally elevated bone regeneration. Wherein 1 is a tissue with abnormal hyperfunction of light bone regeneration, and 2 is a tissue with abnormal hyperfunction of heavy bone regeneration.
FIG. 5 shows the results of in situ immunohistochemical staining of DSG2 in osteochondral tissue with different degrees of abnormally elevated bone regeneration. Wherein, the in situ immunohistochemical staining result of DSG2 in the osteochondral tissue with abnormal and excessive bone regeneration in the first behavior from top to bottom is shown in the 1 st to 4 th diagrams respectively, wherein the 1 st to 4 th diagrams are respectively the partial enlarged view of the first row of the second behavior from top to bottom, wherein the 1 st diagram is the enlarged view of the part shown by the square frame in the 1 st diagram of the first row, the 2 nd diagram is the enlarged view of the part shown by the square frame in the 2 nd diagram of the first row, the 3 rd diagram is the enlarged view of the part shown by the square frame in the 3 rd diagram of the first row, and the 4 th diagram is the enlarged view of the part shown by the square frame in the 4 th diagram of the first row; the degree of hardening of bone tissue increases in order from left to right. The brown yellow color indicates positive expression of DSG2 protein. The boxes represent the enlargement sites.
FIG. 6 shows the results of RT-qPCR assay after endoluminal injection of adeno-associated virus with dsG2 knockdown into a knee joint in an animal model with increased bone regeneration. ". Times" means p <0.05.
Fig. 7 shows the results of safranin O solid-green staining after endoluminal injection of adeno-associated virus that knocks down DSG2 into a knee in an animal model with increased bone regeneration. Wherein, the first line from top to bottom is the safranine O solid green staining of joint parts of mice injected with adeno-associated virus group containing empty plasmid in joint cavities, the second line is the safranine O solid green staining of joint parts of mice injected with shRNA1 and DSG2 knockdown adeno-associated virus group, and the third line is the safranine O solid green staining of joint parts of mice injected with shRNA2 and DSG2 knockdown adeno-associated virus group. The second row from top to bottom corresponds to a partial enlarged view of the first row. The boxes represent the enlargement sites. Wherein, the 1 st image of the second row is the partial enlarged image of the 1 st image of the first row, the 2 nd image of the second row is the partial enlarged image of the 2 nd image of the first row, the 3 rd image of the second row is the partial enlarged image of the 3 rd image of the first row, and the 4 th image of the second row is the partial enlarged image of the 4 th image of the first row.
Fig. 8 is a graph of the Mankin scoring results of fig. 7. "x" means p <0.01.
FIG. 9 is a map of pHBAAV-U6-MCS-CMV-EGFP.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The following examples were run using GRAPHPAD PRISM statistical software on the data, and the experimental results were expressed as mean ± standard deviation, using One-way ANOVA test, P < 0.05 (x) indicated significant differences, P < 0.01 (x) and P < 0.001 (x) indicated very significant differences.
Example 1 Single cell transcriptome sequencing showed that DSG2 expression was up-regulated in osteochondral tissue in patients with abnormally elevated bone regeneration
1. Bioinformatic level DSG2 expression in osteochondral tissue in patients with abnormal hyperactivity of bone regeneration
1. Acquisition of tissue with abnormal hyperfunction of bone regeneration
Discarded mild and severe bone-hardening tissues (patients have signed informed consent) were collected from hospitals.
2. Single cell suspension preparation
Washing 3 times with PBS buffer (containing 0.5% BSA), and shearing the tissue with scissors as much as possible; 1.5 mL RPMI 1640 (Collagenase II, final concentration 2 mg/mL) was added to the 2mL centrifuge tube, and the sample was placed in 37℃for digestion 10 h, during which time the centrifuge tube was spun (12 r.p.m.); placing a 70 μm filter on a 15 mL centrifuge tube, filtering and collecting samples, and respectively cleaning the 70 μm filter and the 2mL centrifuge tube with RPMI 1640 culture medium, wherein the final volume is about 10 mL; centrifugation was performed at 300 g C.for 5 min, the supernatant was completely aspirated, cells were resuspended in 400. Mu.L of PBS, and AO/PI chromatin was examined, and the operations of erythrocyte lysis, dead cell removal, and debris removal were performed depending on the digestion effect.
3.10XGenomics library sequencing
The SINGLE CELL 3' REAGENT KIT x s v2 kit was used for the library construction.
4. Data analysis
The original fastq data was aligned using CELLRANGER (V6.1.2); the sequence of the human transcriptome used was GRCh38-3.0.0 (https:// genome. Ucsc. Edu /); finally obtaining a gene expression matrix;
The original expression matrix was quality controlled using Seurat software (version: 4.1.1) and cells were removed if any of the following conditions were met: filtering cells of less than 3 genes and less than 200 genes; gene expression numbers below 500 and greater than 7500; mitochondrial gene expression ratios were higher than 15%.
The original data is standardized (the standardized mode is LogNormalize, and the scaling factor is 10000); searching for 2000 high variant genes using FindVariableFeatures functions (highly variable features, HVGs); performing unsupervised dimension reduction (30 principal components are selected) on HVGs by PCA (Principal Component Analysis); clustering based on a community clustering (Louvain) algorithm by using FindClusters functions; the data for light and heavy bone sclerosis were analyzed for integration.
After the sub-population is divided, the identified osteochondral tissue stem cells are further classified. Differentially expressed genes of each subpopulation were identified using FINDALLMARKERS functions (DEGs), dividing 6 new subpopulations. Visualization of dimension reduction was performed using DimPlot functions (fig. 1), expression levels of genes in each sample were shown using VlnPlot functions (fig. 2, 4), and expression ranges of genes in UMAP were shown using FeaturePlot functions (fig. 3).
5. Results
FIG. 1 is a graph UMAP showing pooled analysis of osteochondral stem cell populations in tissues with mild and severe abnormally elevated bone regeneration, with a total of 6 cell types identified. FIG. 2 shows the expression of genes involved in hardening in tissues with abnormally elevated light and severe bone regeneration, and the higher the black spot position (position in the picture), the higher the gene expression level. The results showed that osteochondral stem cells present in osteochondral tissue with severe abnormal hyperregeneration had a higher degree of hardening than that of osteochondral tissue with mild abnormal hyperregeneration. Fig. 3 is a population of specifically increased DSG 2-expressing osteochondral stem cells in osteochondral tissue with abnormally high severe bone regeneration, indicating that the population of osteochondral stem cells in osteochondral tissue with severe bone regeneration hyperactivity expresses higher levels of DSG2. Fig. 4 shows the transcriptional expression level of DSG2 in osteochondral tissue with abnormally elevated bone regeneration, and it can be seen that the transcriptional expression level of DSG2 in cells derived from tissue with severe abnormal elevated bone regeneration is increased relative to that of tissue with mild abnormal elevated bone regeneration. The results show that at the bioinformatic level, DSG2 expression is positively correlated with bone sclerosis.
2. Immunohistochemical staining of tissue sections
Knee joint cartilage tissue with abnormal and excessive bone regeneration is taken, fixed in paraformaldehyde, decalcified in 10% EDTA decalcification solution (neutral phosphate buffer as solvent), paraffin embedded and cut into 4 μm slices. The primary antibody, secondary antibody and dilution ratio used for immunohistochemical staining of tissue sections were as follows: DSG2 antibodies (1:100, abcam, ab 96761), HRP-labeled goat anti-rabbit IgG (1:200, servicebio, GB 23303).
The results are shown in FIG. 5, which shows the results of in situ immunohistochemical staining of DSG2 in cartilage tissue of knee joint with abnormally high bone regeneration in the first behavior, from top to bottom, with a partial enlarged view of the first line in the second behavior.
From this, it was found that as the degree of bone sclerosis increased (from left to right in order), the DSG2 staining of cartilage surface cells increased (arrows indicate positive cells). The results showed that with increasing bone sclerosis, the number of DSG2 positive cells increased and that DSG2 expression was positively correlated with the pathological degree of abnormal hyperactivity of bone regeneration.
Example 2 experiment of protection of traumatic osteoarthritis (Posttraumatic osteoarthritis, PTOA) mice joints by dsG2 protein knockdown
1. Construction of DSG2 recombinant vector
1. Carrier information
The interfering vector pHBAAV-U6-MCS-CMV-EGFP (Hantao Biotechnology (Shanghai) Co., ltd.) was selected and a map described in non-patent document "Junliang Kuang, Jieyi Wang, Yitao Li, Mengci Li, Mingliang Zhao, Kun Ge, Dan Zheng, Kenneth C P Cheung, Boya Liao, Shouli Wang, Tianlu Chen, Yinan Zhang, Congrong Wang, Guang Ji, Peng Chen, Hongwei Zhou, Cen Xie, Aihua Zhao, Weiping Jia, Xiaojiao Zheng, Wei Jia. Hyodeoxycholic acid alleviates non-alcoholic fatty liver disease through modulating the gut-liver axis. Cell Metab. 2023 Oct 3;35(10):1752-1766.e8. doi: 10.1016/j.cmet.2023.07.011."） is shown in FIG. 9. PAAV-RC vector plasmid and pHelper vector plasmid were derived from Hantao Biotechnology (Shanghai) Co., ltd.
2. Interference target design and primer synthesis
The CDS region of the DSG2 gene is SEQ ID No.2. According to SEQ ID No.2, siRNA was designed and designated shRNA1 and shRNA2. The siRNA produced by shRNA1 targets the 1020 th to 1044 th bases of SEQ ID No.2. The siRNA produced by shRNA2 targets 1096 to 1120 bases of SEQ ID No.2.
DSG2 protein coding gene siRNA sequence and shRNA sequence:
shRNA1-F（SEQ ID No.7）：AATTCGccttgtcaaggaagtggactatgaaTTCAAGAGAttcatagtccacttccttgacaaggTTTTTTG
shRNA1-R（SEQ ID No.8）：GATCCAAAAAAccttgtcaaggaagtggactatgaaTCTCTTGAAttcatagtccacttccttgacaaggCG
shRNA2-F（SEQ ID No.9）：AATTCGcacaagtccattctgagcaagtacaTTCAAGAGAtgtacttgctcagaatggacttgtgTTTTTTG
shRNA2-R（SEQ ID No.10）：GATCCAAAAAAcacaagtccattctgagcaagtacaTCTCTTGAAtgtacttgctcagaatggacttgtgCG
control viral vector shRNA-F (SEQ ID No. 11): GATCCGTTCTCCGAACGTGTCACGTAATTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTC A
Control viral vector shRNA-R (SEQ ID No. 12): AATTGAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGAATTACGTGACACGTTCGGAGAACG A
The lower case letters are used as target points.
The primers were oligo sequences purified by biological company synthetic PAGE, and each was diluted to 100uM. The annealing product was obtained after the treatment according to the following system and annealing procedure, respectively. The system is as follows (20 uL): 10*oligo Buffer 2. Mu.L, primer-F1. Mu.L, primer-R1. Mu.L, H 2 O make up 20. Mu.L. Annealing procedure, 95℃for 10min, 75℃for 10min, 55℃for 10min, 35℃for 10min, 15℃for 10 min.
3. And (3) carrier enzyme cutting: adding each reagent according to the following enzyme digestion system, gently sucking and beating, uniformly mixing, placing in a water bath kettle at 37 ℃ for reaction of 1-2 h, performing agarose gel electrophoresis after enzyme digestion is finished, and recovering the target fragment to obtain the carrier after enzyme digestion. And (3) enzyme cutting system: pHBAAV-U6-MCS-CMV-EGFP vector DNA (1. Mu.g/. Mu.L) 1. Mu.L, 10 Xbuffer 4. Mu.L, ddH 2 O32. Mu.L, ecoRI 1.5. Mu.L, bamHI 1.5. Mu.L.
4. Interference fragment and carrier linking
The connection system is as follows: 4 mu L of the annealed product in the step 2, more than ng of the carrier after enzyme digestion in the step 3, 1 mu L of T4 connecting buffer and 20 mu L of T4 connecting enzyme are complemented by water. The above-mentioned connection system was connected at 22℃for 1-2 h or at 16℃for overnight.
5. Transformation
1) After DH5 alpha competent cells are taken out from the refrigerator at the temperature of minus 80 ℃, the cells are immediately put on ice for melting, and the competent split charging process is operated gently, so that the mechanical damage to the cells is reduced;
2) After melting of competence, split charging in a volume of 50. Mu.L per tube (20. Mu.L is enough for plasmid transformation), adding 5. Mu.L of ligation product, and standing on ice for 20-30min;
3) Heat shock 90 s at 42 ℃, and inserting 2-3 min on ice immediately after heat shock;
adding 500 mu L of LB culture medium into an ultra clean bench, and gently reversing the culture medium for 3 to 5 times;
4) Shake culturing at 37deg.C and 230rpm to 45-60 min;
5) The bacterial liquid is coated on a solid flat plate with corresponding resistance, the coating is uniform, and then the plate is placed in a constant temperature oven with the temperature of 37 ℃ for culturing for 12-16h.
6. The monoclonal was picked for sequencing.
The sequencing result shows that the fragment (small fragment) between EcoRI and BamHI recognition sites of pHBAAV-U6-MCS-CMV-EGFP is replaced by a DNA molecule formed by annealing shRNA1-F and shRNA1-R, and a recombinant expression vector obtained by keeping other sequences of pHBAAV-U6-MCS-CMV-EGFP unchanged is named HBAAV/5-m-Dsg 2 shRNA1-EGFP (interference vector). The DNA molecule formed by annealing shRNA1-F and shRNA1-R codes shRNA taking a mouse DSG2 gene as a target point, the shRNA generates siRNA interfering with the mouse DSG2 gene, one strand sequence of the siRNA is 5'-ccuugucaaggaaguggacuaugaa-3' (SEQ ID No. 3), and the other strand sequence of the siRNA is 5'-uucauaguccacuuccuugacaagg-3' (SEQ ID No. 4).
The sequencing result shows that the fragment (small fragment) between EcoRI and BamHI recognition sites of pHBAAV-U6-MCS-CMV-EGFP is replaced by a DNA molecule formed by annealing shRNA2-F and shRNA2-R, and a recombinant expression vector obtained by keeping other sequences of pHBAAV-U6-MCS-CMV-EGFP unchanged is named HBAAV/5-m-Dsg 2 shRNA2-EGFP (interference vector). The DNA molecule formed by annealing shRNA2-F and shRNA2-R codes shRNA taking a mouse DSG2 gene as a target point, the shRNA generates siRNA interfering with the mouse DSG2 gene, one strand sequence of the siRNA is 5'-cacaaguccauucugagcaaguaca-3' (SEQ ID No. 5), and the other strand sequence of the siRNA is 5'-uguacuugcucagaauggacuugug-3' (SEQ ID No. 6).
The sequencing result shows that the fragment (small fragment) between EcoRI and BamHI recognition sites of pHBAAV-U6-MCS-CMV-EGFP is replaced by a DNA molecule formed by annealing a control viral vector shRNA-F and a control viral vector shRNA-R, and the recombinant expression vector obtained by keeping other sequences of pHBAAV-U6-MCS-CMV-EGFP unchanged is named as the control viral vector shRNA. The DNA molecule encodes a non-target shRNA, which is designated NC-shRNA.
2. Preparation of recombinant adeno-associated virus with DSG2 knockdown
Preparation of shRNA1 and dsG2 knockdown adeno-associated virus: and co-transfecting HBAAV/5-m-Dsg 2 shRNA1-EGFP, pAAV-RC vector plasmid and pHelper vector plasmid into 293T cells, harvesting cell precipitates at 72 h after transfection, and obtaining the high-titer shRNA 1-knockdown DSG2 adeno-associated virus preservation solution by adopting a column purification mode.
Preparation of shRNA 2-knocked-down DSG2 adeno-associated virus: and co-transfecting HBAAV/5-m-Dsg 2 shRNA2-EGFP, pAAV-RC vector plasmid and pHelper vector plasmid into 293T cells, harvesting cell precipitates at 72 h after transfection, and obtaining the high-titer shRNA 2-knockdown DSG2 adeno-associated virus preservation solution by adopting a column purification mode.
Adeno-associated virus preparation containing empty plasmid (empty vector): and (3) replacing HBAAV/5-m-Dsg 2 shRNA1-EGFP with pHBAAV-U6-MCS-CMV-EGFP vector empty plasmid, and carrying out the rest operation steps of the adeno-associated virus with the shRNA1 knockdown DSG2 to obtain the adeno-associated virus preservation solution containing empty plasmid.
3. Construction of mouse PTOA model
A model of traumatic osteoarthritis in mice was constructed by anterior cruciate ligament transection (Anterior Cruciate Ligament Transection, ACLT) surgery. 8 week old C57BL/6 mice (purchased from Vetolihua, cat# strain code 213) were selected and ACLT operated on their knees. Abdominal injection of pentobarbital sodium was used for anesthesia, hair around the knee joint was shaved off, the mouse was fixed on an operating table, and the knee joint of the mouse was rubbed with 75% alcohol for sterilization. Skin at the knee joint of the mouse is scratched by a surgical knife, the knee joint is exposed, the anterior cruciate ligament is separated vertically downwards from the outer side of the patellar ligament, whether molding is successful (PTOA mouse) is judged through a drawer test, and skin is sutured after operation, and the device is wiped and disinfected by iodophor.
After molding 1w, 10 μl of adeno-associated virus (1.2X10 12 vg/mL) containing empty plasmid or shRNA1 DSG2 knockdown adeno-associated virus (1.6X10 12 vg/mL) or shRNA2 DSG2 knockdown adeno-associated virus (1.4X10 12 vg/mL) was injected into the joint cavity of the PTOA mice by microinjectors, respectively, to obtain joint local DSG2 knockdown PTOA mouse model 1 (shRNA 1), joint local DSG2 knockdown PTOA mouse model 2 (shRNA 2) and control PTOA mouse model (Vector, injected with adeno-associated virus containing empty plasmid).
4. Knock-down efficiency validation of DSG2 adeno-associated virus
(1) 4W after ACLT operation, euthanize the mice, collect the complete knee joint of the mice, remove soft tissues such as muscle and ligament, fix 48 h in 4% paraformaldehyde, decalcify in 10% EDTA decalcification solution (the solvent is neutral phosphate buffer solution) for 2 weeks, then carry out paraffin embedding, cut into 4 μm slices.
(2) ShRNA group 1: total RNA of joint tissue of PTOA mouse model 1 (shRNA 1) with local DSG2 knockdown of joints was extracted using tissue RNA extraction kit (ES SCIENCE, RN002 plus). cDNA was synthesized by reverse transcription using 20. Mu.L of a reverse transcription system (Servicebio, G3337), and fluorescence quantitative PCR was performed using SYBR GREEN QPCR MASTER Mix (Servicebio, G3326), apparatus model ABI 7500 (Applied Biosystems). The expression quantity of Dsg2 in the knee joint of the mouse is detected and evaluated by an RT-qPCR experiment, and the knock-down efficiency of the Dsg2 is evaluated by internal reference of Gapdh gene. Experiments were repeated 5 times with 1 mouse at a time.
And analyzing the RT-qPCR result by adopting △△ Ct method, and calculating the relative expression quantity of the genes. Test data were processed using GRAPHPAD PRISM and significance analyzed using IBM SPSS STATISTICS 22 statistical software. Comparison between the two sets of data was checked using Student's t; the comparison between three or more data uses Duncan's multiple comparisons to perform one-way analysis of variance. p <0.05 indicates significant differences, p <0.01 indicates very significant differences, and p <0.001 indicates very significant differences.
Dsg2-F（SEQ ID No.13）：5'-GGAAACGGACTTCACTTAGAGG-3'；
Dsg2-R（SEQ ID No.14）：5'-TGGCAATCGGGTTCTTTCTGG-3'；
Gapdh-F（SEQ ID No.15）：5'-CGGTGCTGAGTATGTCGTGGAGTCT-3'；
Gapdh-R（SEQ ID No.16）：5'-GCTAAGCAGTTGGTGGTGCAGGATG-3'。
ShRNA2 group: the PTOA mouse model 2 (shRNA 2) with the local DSG2 knockdown joint is used for replacing the PTOA mouse model 1 (shRNA 1) with the local DSG2 knockdown joint of the shRNA1 group, and the rest operations are the same as the shRNA1 group.
Vector group: the control PTOA mouse model is used for replacing the PTOA mouse model 1 (shRNA 1) with the shRNA1 group with locally reduced joint DSG2, and the rest operations are the same as the shRNA1 group.
The relative expression levels of the DSG2 gene are shown in FIG. 6. The DSG2 content in joint tissue of mice in shRNA 1-2 group was significantly lower than in empty vector group (P < 0.05).
6. Pathological staining of tissue sections
Tissue morphology changes were observed by safranin O-fast green (SOFG) staining. The Mankin Score (Mankin Score) statistical method for safranin O-fast green (SOFG) stained pictures is as follows:
① Score evaluation rule for cartilage structure: normal 0 minutes, 1 minute of surface irregularity, 2 minutes of pannus formation and surface irregularity, 3 minutes of cracks entering the transition layer, 4 minutes of cracks entering the radiation layer, 5 minutes of cracks entering the calcification layer, and 6 minutes of complete structural damage;
② Score evaluation rules for chondrocytes: normal 0 score, diffuse cells increased by 1 score, local cells increased by 2 scores, and cell numbers significantly decreased by 3 scores;
③ Scoring criteria for cartilage matrix staining: normal 0 score, slight 1 score decrease, moderate 2 score decrease, severe 3 score decrease, no coloration 4 score;
④ Score evaluation rules for tidal line integrity: complete 0 score, 1 score destroyed by blood vessel;
the sum of the 4 items is Mankin scoring result, and the higher the score, the more serious the cartilage degeneration degree.
The results of safranin O-fast green (SOFG) staining are shown in FIG. 7, in which FIG. 7, from top to bottom, FIG. 1, the safranin O solid green staining of joint parts of mice injected with adeno-associated virus group containing empty plasmid, FIG. 2, the safranin O solid green staining of joint parts of mice injected with adeno-associated virus group with shRNA1 knockdown DSG2, and FIG. 3, the safranin O solid green staining of joint parts of mice injected with adeno-associated virus group with shRNA2 knockdown DSG 2. The second row from top to bottom corresponds to a partial enlarged view of the first row. The NC Vector group joint surface is uneven, the cartilage layer is thinned, and the cartilage matrix is reduced; compared with NC Vector group, cartilage matrix is increased and joint surface is flat in mice with DSG2 protein knockdown.
The Mankin score calculated from the safranin O-fast green (SOFG) staining results in FIG. 7 is shown in FIG. 8, where the Mankin score was significantly reduced in the DSG2 protein knockdown group of mice in FIG. 8. DSG2 protein knockdown inhibited to some extent the pathological hyperactivity of bone regeneration in traumatic osteoarthritis mice.
The sequences referred to in the above examples:
amino acid sequence of DSG2 （SEQ ID No.1）MARSPGDRCALLLLVQLLAVVCLDFGNGLHLEVFSPRNEGKPFPKHTHLVRQKRAWITAPVALREGEDLSRKNPIAKIHSDLAEEKGIKITYKYTGKGITEPPFGIFVFDRNTGELNITSILDREETPYFLLTGYALDSRGNNLEKPLELRIKVLDINDNEPVFTQEVFVGSIEELSAAHTLVMKITATDADDPETLNAKVSYRIVSQEPANSHMFYLNKDTGEIYTTSFTLDREEHSSYSLTVEARDGNGQITDKPVQQAQVQIRILDVNDNIPVVENKMYEGTVEENQVNVEVMRIKVTDADEVGSDNWLANFTFASGNEGGYFHIETDTQTNEGIVTLVKEVDYEEMKKLDLSIIVTNKAAFHKSILSKYKATPIPITVKVKNVVEGIHFKSSVVSFRASEAMDRSSLSRSIGNFQVFDEDTGQAAKVTYVKVQDTDNWVSVDSVTSEIKLVKIPDFESRYVQNGTYTAKVVAISKEHPQKTITGTIVITVEDVNDNCPVLVDSVRSVCEDEPYVNVTAEDLDGAQNSAPFSFSIIDQPPGTAQKWKITHQESTSVLLQQSERKRGRSEIPFLISDSQGFSCPERQVLQLTVCECLKGGGCVAAQYDNYVGLGPAAIALMILALLLLLLVPLLLLICHCGGGAKGFTPIPGTIEMLHPWNNEGAPPEDKVVPSLLVADHAESSAVRGGVGGAMLKEGMMKGSSSASVTKGQHELSEVDGRWEEHRSLLTAGATHHVRTAGTIAANEAVRTRATGSSRDMSGARGAVAVNEEFLRSYFTEKAASYNGEDDLHMAKDCLLVYSQEDTASLRGSVGCCSFIEGELDDLFLDDLGLKFKTLAEVCLGRKIDLDVDIEQRQKPVREASVSAASGSHYEQAVTSSESAYSSNTGFPAPKPLHEVHTEKVTQEIVTESSVSSRQSQKVVPPPDPVASGNIIVTETSYAKGSAVPPSTVLLAPRQPQSLIVTERVYAPTSTLVDQHYANEEKVLVTERVIQPNGGIPKPLEVTQHLKDAQYVMVRERESILAPSSGVQPTLAMPSVAAGGQNVTVTERILTPASTLQSSYQIPSETSITARNTVLSSVGSIGPLPNLDLEESDRPNSTITTSSTRVTKHSTMQHSYS.
2. DSG2 coding region sequence (SEQ ID No. 2)
5'atggcgcggagcccgggtgaccggtgcgccctgctgctgctggtgcagctgctggcggtggtctgcttggactttggaaacggacttcacttagaggtcttcagcccaagaaatgaaggcaaaccgttccctaagcacactcacttggttcgtcaaaagagggcctggatcactgcccctgtggctctgcgggagggcgaagacctgtccagaaagaacccgattgccaagatacactctgaccttgcagaagaaaaagggataaaaatcacgtacaagtacactgggaagggaattacagaaccgcctttcggcatattcgtctttgatagaaacacaggagaactgaacatcactagcattcttgaccgggaagaaacaccatattttctgctgacaggctatgcattggactccagaggaaacaacctggaaaagcccttggaactacgcatcaaagttctggacatcaatgacaacgagccagtgttcacacaggaggtctttgttgggtccattgaggaattgagtgcagcacatacacttgtgatgaaaatcaccgccacagatgcagatgacccggagactctgaatgctaaagtctcctacagaattgtctctcaggagcctgcaaatagtcatatgttctacctaaataaagacacgggggagatctatacgaccagttttactttggacagagaggaacacagcagctattccttgacggtggaagcaagagatggtaacgggcagataacagacaagccagtccagcaagctcaagttcagatccgtatattggatgtcaatgacaatatacctgtggtagaaaacaaaatgtatgaggggacagtggaagaaaaccaggtcaatgtagaagtcatgcggatcaaagtgaccgatgcagatgaagtgggctctgataactggctagcaaactttacatttgcatcaggaaatgaagggggctatttccacattgagactgacacacagactaatgaagggattgtgacccttgtcaaggaagtggactatgaagaaatgaagaagctagacttgagcatcattgtcactaacaaagcagctttccacaagtccattctgagcaagtacaaggccacgcccattcccatcactgtcaaggtcaagaacgtggttgaaggcattcatttcaagagcagcgtagtctctttccgagctagtgaggcaatggatagatccagcctcagcaggtcgattggaaattttcaagtttttgatgaagacactggtcaagcagctaaagtaacatatgtaaaagtgcaagacactgacaactgggtctctgtggactccgtcacttcagagattaagcttgtaaagattcctgactttgaatctagatatgtccaaaatggtacctacactgcaaaggttgtggccatatccaaagaacatcctcaaaaaaccatcactggcaccatcgttatcactgttgaagacgtcaatgacaattgtcccgtgctggtggactctgtacggagtgtctgtgaggatgaaccatatgtgaatgtcactgcagaggatttggatggggcccagaacagtgcgccattcagcttctccatcattgaccagcctcctggaacggcacagaagtggaaaataacgcaccaggaaagtaccagtgtgctgctgcagcagagcgagcggaaacgcgggagaagtgagattcccttcctcatttccgacagccagggcttcagctgccccgaaaggcaggtccttcagctcactgtatgcgagtgtctgaagggcggtggctgtgtggctgcacagtatgacaactacgtcgggttgggccctgccgccatcgctctcatgattctagcactcctgctcctgctcctggtgccgctcttgctgttgatatgccactgtggagggggcgccaaaggcttcacccccattcctgggacaatagagatgctgcacccttggaataatgaaggggcacctcctgaggacaaggtggtgccatcgcttctggtggccgatcatgcagagagctcggcagtgagaggcggcgtaggaggtgcgatgctcaaggaaggcatgatgaaaggcagcagctcagcttccgttaccaaagggcagcatgagctgtctgaggttgacggaagatgggaagaacacagaagcctcctcaccgctggggccactcaccatgtaaggacagcaggaaccatcgctgccaacgaagccgtaaggacaagagccacggggtcttccagagacatgagtggggctcgaggagccgttgccgtgaatgaggaattcttaagaagttacttcacagagaaagcggcctcctacaatggggaagacgaccttcacatggccaaagactgccttctcgtttactctcaggaagacacggcctccctccgaggctcggtcgggtgctgcagtttcatcgagggagaactcgatgacctgttcctggatgatcttggccttaaattcaagaccctagctgaagtttgcctaggtcgaaagatcgatctggatgtggacattgaacagaggcagaagccggtcagagaagcgagcgtgagtgcagcttctggctcgcactatgagcaagcggtaaccagctcagagagcgcgtactcctctaacaccggcttccccgcccccaaacctctgcacgaagtgcacacagagaaagtcacacaggaaatcgtcactgagagctctgtatcttccaggcagagtcagaaggtagtaccgccacctgatcctgtggcttctggtaatattatagtgacggaaacttcctatgccaaaggctcagcagtgccacccagcactgtgctcctggctcccagacagccacagagcctgatcgtgacagagagggtgtatgctccaacctccaccttggtggatcagcattatgccaatgaagaaaaagtccttgttaccgaacgagtgatccagcctaatgggggcatccctaagccccttgaggtcacccagcatctgaaagatgcacagtatgtaatggtgagggaaagagagagcatccttgctcccagctcaggcgtgcagcccactctggcaatgcccagcgtggcagcaggaggacagaatgtcaccgtgacagaaagaatactaactcctgcttccactctgcagtccagctaccagattcccagtgaaacctccatcacggctaggaacactgtgctctctagtgtgggaagcataggtcctctgcccaatttagatctagaggaatctgatcgtcccaattctactataaccacatcttccaccagggtcaccaagcatagcaccatgcaacattcttactcctaa-3'.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (9)
1. Use of a biomaterial in the manufacture of a medicament for inhibiting pathological hyperactivity of bone regeneration, characterized in that the biomaterial is any of the following:
b1 An RNA molecule that inhibits or reduces or down-regulates expression of a gene encoding desmoglein 2 or a chemical modification of said RNA molecule;
B2 A gene encoding the RNA molecule of B1);
B3 An expression cassette comprising the gene of B2);
b4 A recombinant vector comprising the gene of B2), or a recombinant vector comprising the expression cassette of B3);
b5 A recombinant microorganism comprising the gene of B2), a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4);
B6 A transgenic animal tissue comprising B1) said RNA molecule, or a transgenic animal tissue comprising B2) said expression cassette;
b7 A transgenic animal organ containing the RNA molecule of B1), or a transgenic animal organ containing the expression cassette of B2).
2. The use according to claim 1, wherein B1) the RNA molecule targets mRNA transcribed from the gene encoding desmoglein 2.
3. The use according to claim 2, wherein the desmoglein 2 encoding gene is SEQ ID No.2.
4. A medicament for inhibiting pathological hyperfunction of bone regeneration, which is characterized by comprising medicinal auxiliary materials and substances for down-regulating encoding genes of desmoglein 2.
5. The medicament according to claim 4, wherein the substance is any one of the following:
b1 An RNA molecule that inhibits or reduces or down-regulates expression of the coding gene or a chemical modification of the RNA molecule;
B2 A gene encoding the RNA molecule of B1);
B3 An expression cassette comprising the gene of B2);
b4 A recombinant vector comprising the gene of B2), or a recombinant vector comprising the expression cassette of B3);
B5 A recombinant microorganism comprising the gene of B2), a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4).
6. The drug of claim 5, wherein the RNA molecule targets mRNA transcribed from the gene encoding desmoglein 2.
7. The drug according to claim 5, wherein the desmoglein 2 encoding gene is SEQ ID No.2.
8. The drug of claim 5, wherein the RNA molecule is an siRNA, and the siRNA is SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or/and SEQ ID No.6.
9. The medicament of claim 4, wherein the pathological hyperfunction of bone regeneration comprises pathological bone regeneration caused by one or more of osteoarthritis, ectopic ossification, spinal fusion.
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