COMPOSITION FOR DIAGNOSING MUSCULOSKELETAL DISEASES, COMPOSITION FOR PREVENTING OR TREATING MUSCULOSKELETAL DISEASES, AND USE THEREOF
20230235402 · 2023-07-27
Assignee
- Seoul National University Hospital (Seoul, KR)
- Seoul National University R&DB Foundation (Seoul, KR)
- CHEONGJU UNIVERSITY INDUSTRY & ACADEMY COOPERATION FOUNDATION (Cheongju-si Chungcheongbuk-do, KR)
Inventors
- Hyun Chul JO (Seoul, KR)
- Young-il HWANG (Seoul, KR)
- Jin-Hee KIM (Cheongju-si Chungcheongbuk-do, KR)
- Jin-Hong KIM (Seoul, KR)
- Jae-Hyung LEE (Seoul, KR)
- Hyun-Ju LIM (Jeonju-si Jeollabuk-do, KR)
- Ah-Young LEE (Anyang-si Gyeonggi-do, KR)
- Seung Yeon LEE (Seoul, KR)
- Ji-Hye YEA (Hwaseong-si Gyeonggi-do, KR)
- Yeasol KIM (Seoul, KR)
Cpc classification
A61P21/00
HUMAN NECESSITIES
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
C12Q1/6883
CHEMISTRY; METALLURGY
Abstract
A composition suitable for diagnosing a musculoskeletal disease and a composition suitable for preventing or treating a musculoskeletal disease are disclosed. The composition contains zinc finger protein with KRAB and SCAN domains 8 (Zkscan8) protein, which can be effectively used as an excellent biomarker for obtaining accurate information about the occurrence and progression stages of a musculoskeletal disease, specifically a tendon disease or a ligament disease. The compositions containing Zkscan8 can be effectively used for preventing or treating a musculoskeletal disease through Zkscan8 overexpression.
Claims
1. A composition for diagnosing a musculoskeletal disease, comprising a Zkscan8 (zinc finger protein with KRAB and SCAN domains 8) gene or an agent for measuring the expression level of a protein encoded by the gene as an active ingredient.
2. The composition according to claim 1, wherein the agent is a primer or a probe binding specifically to the Zkscan8 gene.
3. The composition according to claim 1, wherein the agent is an antibody or an aptamer binding specifically to a protein encoded by the Zkscan8 gene.
4. The composition according to claim 1, wherein the musculoskeletal disease is a tendon disease or a ligament disease.
5. A method for providing information necessary for diagnosing a musculoskeletal disease, comprising: 1) a step of measuring the expression level of a Zkscan8 (zinc finger protein with KRAB and SCAN domains 8) gene or a protein encoded by the gene in a biological sample isolated from a subject suspected of a musculoskeletal disease; and 2) a step of comparing the measured expression level of the Zkscan8 gene or the protein encoded by the gene with the expression level of a normal control group sample.
6. A composition for preventing or treating a musculoskeletal disease, comprising a Zkscan8 (zinc finger protein with KRAB and SCAN domains 8) protein as an active ingredient.
7. The composition according to claim 6, wherein the Zkscan8 protein has an amino acid sequence represented by SEQ ID NO 2.
8. The composition according to claim 6, wherein the musculoskeletal disease is a tendon disease or a ligament disease.
9. The composition according to claim 8, wherein the tendon disease is one or more selected from a group consisting of tendon injury, tendon rupture, tendinitis, tendinosis and tendosynovitis, and the ligament disease is one or more selected from a group consisting of knee quadriceps ligament rupture, coronary ligament sprain, collateral ligament rupture, anterior cruciate ligament injury, posterior cruciate ligament injury, ankle ligament injury, ligament rupture and sprain.
10. A pharmaceutical composition for preventing or treating a musculoskeletal disease, comprising a Zkscan8 (zinc finger protein with KRAB and SCAN domains 8) gene, a vector comprising the gene, a cell comprising the vector or a culture thereof as an active ingredient.
11. The composition according to claim 10, wherein the vector is a linear DNA, a plasmid DNA or a recombinant viral vector.
12. The composition according to claim 11, wherein the recombinant virus is any one selected from a group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and lentivirus.
13. The composition according to claim 10, wherein the cell is one or more selected from a group consisting of a stem cell, a dendritic cell, an autologous tumor cell and an established tumor cell.
14. The composition according to claim 13, wherein the stem cell is a mesenchymal stem cell.
15. The composition according to claim 14, wherein the mesenchymal stem cell is an umbilical cord-derived mesenchymal stem cell.
16. The composition according to claim 10, wherein the musculoskeletal disease is a tendon disease or a ligament disease.
17. The composition according to claim 16, wherein the tendon disease is one or more selected from a group consisting of tendon injury, tendon rupture, tendinitis, tendinosis and tendosynovitis, and the ligament disease is one or more selected from a group consisting of knee quadriceps ligament rupture, coronary ligament sprain, collateral ligament rupture, anterior cruciate ligament injury, posterior cruciate ligament injury, ankle ligament injury, ligament rupture and sprain.
18. The composition according to claim 10, wherein the composition increases the expression or activity of Scx (scleraxis), Mkx (mohawk), Egr-1 (early growth response-1) and Egr-2 (early growth response-2).
19. A method for screening a composition for preventing or treating a musculoskeletal disease, comprising: (a) a step of contacting injured tendon, ligament, muscle, cartilage or bone cells with a test substance; and (b) a step of measuring the expression level of a Zkscan8 (zinc finger protein with KRAB and SCAN domains 8) gene or protein in the sample, wherein, if the expression level of the Zkscan8 is increased as compared to a normal control group in the step (b), the test substance is determined as a composition for preventing or treating a musculoskeletal disease.
20. The method according to claim 19, wherein the musculoskeletal disease is a tendon disease or a ligament disease.
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE
[0125] Hereinafter, the present disclosure will be described in more detail through examples. The examples are provided only to describe the present disclosure more specifically and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.
Examples
[0126] Experimental Methods
[0127] Screening and Selection of Genes
[0128] In order to search for biomarkers related with musculoskeletal diseases, whole transcriptome expression profiling microarray datasets GSE30138 and GSE54207 and RNA sequencing dataset GSE65180 associated with mouse limb tenogenesis were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.gov/geo/) of the National Center for Biotechnology Information (NCBI) and analyzed for screening of tendon regeneration candidate genes.
[0129] For GSE30138 and GSE54207, the GPL1261 platform (Affymetrix mouse genome 430 2.0 array) was used. And, for GSE65180, the GPL13112 platform [Illumina Hiseq 2000 (Mus musculus)] was used. Genes showing similar expression patterns depending on embryonic stages as tenogenesis markers (Tgf-β2, Scx, Mkx, Egr1, Tnmd, Thbs4, Col1, Gag, Tnc), myogenesis markers (Pax3, Pax7, Myf5, Des, MyoD, myogenin, MRF4, myostatin, MHC2), osteogenesis marker (Runx2, Osterix, Alp, Ocn, Opn) and chondrogenesis markers (Comp, Sox9, Chad, Acan, Col2a1) were screened primarily.
[0130] For construction of the protein-protein interaction (PPI) network and investigation of the interaction between genes, analysis was conducted using the TiCoNE and ClueGo plugins of the Cytoscape software (http://www.cytoscape.org/) (
[0131] RNA-Seq Analysis
[0132] Total RNAs of a normal tendon (n=5) and a rotator cuff (n=6) were extracted using an RNeasy Plus universal kit (Cat. 73404. Qiagen, USA) according to the manufacturer's protocol. The integrity of the total RNAs was identified with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) and a cDNA library was created using a TruSeq stranded mRNA library prep kit (Cat. 20020594; Illumina, USA) according to the manufacturer's protocol. The pooled library was loaded on HiSeq 2000 at a concentration of 2 nM and then sequenced (v3 SBS chemistry, 76 cycles, paired-end).
[0133] Culturing of Human Umbilical Cord-Derived Stem Cells and Tendon Cells
[0134] All the tissues used in the study were acquired and used under the consent of patients. Umbilical cord and tendon tissues were washed 2-3 times with Ca.sup.2+- and Mg.sup.2+-free Dulbecco's phosphate-buffered saline (DPBS, GIBCO, NY, USA) supplemented with antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin sulfate and 0.25 μg/mL amphotericin B (antibiotic-antimycotic solution; Welgene, Daegu, Korea)) in order to remove impurities such as blood, etc.
[0135] Umbilical cord was acquired from a patient who received Caesarean section. After measuring length and weight, the umbilical cord was cut to about 2-4 mm in length using surgical scissors, and an amount corresponding to 1 g was aligned and inoculated onto a 150 cm.sup.2 culture dish. After the umbilical cord completely adhered to the culture dish, it was cultured using a culture medium (LG DMEM, 10% fetal bovine serum (FBS; Welgene, Daegu, Korea) and antibiotic-antimycotic solution) at 37° C. while supplying 5% CO.sup.2.
[0136] Tendon tissues obtained from a patient with complete rotator cuff tear were cultured at 37° C. for 2 hours with light shaking in HG DMEM (high-glucose Dulbecco's modified Eagle's medium, Welgene, Daegu, Korea) supplemented with 0.3% type 2 collagenase (GIBCO) and antibiotics. Subsequently, after adding a culture medium (HG DMEM, 10% FBS) and antibiotics (antibiotic-antimycotic solution) of the same volume, undissociated tissues were removed using a 100-μm cell filter. Cells recovered by centrifuging (500 g, 15 minutes) at 20° C. were washed twice with a culture medium. The recovered cells were counted by trypan blue exclusion assay. The cells were transferred onto a culture dish at a density of 2×10.sup.4 to 5×10.sup.4 cells/cm.sup.2 and then cultured at 37° C. in a 5% CO.sup.2 incubator.
[0137] When the cells grew to fill about 60-80% of the culture dish, they were washed twice with DPBS and adherent cells were detached by treating with trypsin-EDTA (Welgene, Daegu, Korea) containing 0.05% trypsin and 0.53 mM EDTA (ethylenediaminetetraacetic acid) for 3 minutes. Then, the cells were stained and counted by trypan blue exclusion assay. Human umbilical cord-derived stem cells (3,333 cells/cm.sup.2) and tendon cells were cultured continuously at a ratio of 1:3.
[0138] Preparation of Zkscan8-Introduced Umbilical Cord-Derived Mesenchymal Stem Cells (MSC-Zk8)
[0139] A pscAAV-GFP vector plasmid (Cell Biolabs, Calif., USA) was used as a control group vector. After cleaving the GFP region with restriction enzymes BamHI and Sall, Zkscan8 was cloned into the region using primers (FP: 5′-AAGGATCCATGTACCCATACGATGTTCCAGATTACGCTATGGCGGAGGAAAGTC GG-3′, RP: 5′-AAGTCGACCTAGACTGAGATAGACTC-3′) and BamHI and Sall restriction enzymes. The cleavage map of the pscAAV-Zkscan8 vector is shown in
[0140] Analysis of Proliferation Ability
[0141] Proliferation ability was analyzed by measuring the amount of living cells by WST assay. After inoculating the gene-introduced umbilical cord-derived mesenchymal stem cells onto a culture dish at a density of 5×10.sup.4 cells/cm.sup.2, 10 μL of Ez-Cytox (DoGEN, Guro, Korea) was added to the culture medium on days 3, 5, 9, 12 and 15. After incubation at 37° C. in a 5% CO.sup.2 incubator for 1 hour, absorbance was measured at a wavelength of 450 nm.
[0142] Differentiation of Umbilical Cord-Derived Mesenchymal Stem Cells into Tendon
[0143] The umbilical cord-derived mesenchymal stem cells (UCMSCs) were inoculated onto a culture dish at a density of 3×10.sup.4/cm.sup.2 in a DMEM-HG medium containing 10% FBS. 2 days later, the medium was replaced with a 1% FBS culture medium. Next day, the medium was replaced with a DMEM-HG medium containing 10% FBS and supplemented with 50 μg/mL ascorbic acid and 10 ng/mL connective tissue growth factor (CTGF) and differentiation was induced for 3, 7 or 14 days.
[0144] For fibrinogen-based 3D culture, Sylgard (Dow Corning, Midland, Mich., USA) was coated on a 35-mm culture dish for 1-2 weeks and then two silk sutures of 5 mm in length were pinned on the Sylgard. After sterilizing the culture dish and sutures on a clean bench using 100% ethanol and UV and drying for 2 hours, they were immersed in DMEM for 1 hour at 37° C. in a CO.sub.2 incubator. After mixing the gene-introduced MSCs and UCMSCs (2.25×10.sup.5 and 0.75×10.sup.5 cells respectively) at a ratio of 3:1 with a thrombin mixture (MEM supplemented with 10% fetal bovine serum, 1% antibiotic solution, 1 U/mL thrombin, 200 μM aminocaproic acid and 10 mg/mL aprotinin), a 10 mg/mL fibrinogen solution was added. After spreading the mixture on the Sylgard-coated culture dish quickly, it was left at 37° C. for 3 hours. Then, the cells were cultured for 3 days in a tendon cell differentiation medium (DMEM-HG containing 250 μM ascorbic acid and 50 μM L-proline). In order to investigate differentiation into tendon cells, the cells were stained with hematoxylin and eosin (H&E) and picrosirius red and subjected to immunohistochemical staining. In addition, the degree of differentiation was investigated from the expression of tendon cell marker genes. After fixing the cells in a 4% paraformaldehyde solution for 24 hours, 6-μm section samples were prepared by embedding in paraffin. Then, they were stained with H&E and picrosirius red. They were immunohistochemically stained by reacting with anti-Col I (ab34710, Abcam) and anti-Col III (ab7778, Abcam) diluted to 1:200 for a day in a refrigerator, followed by reaction with HRP-conjugated secondary antibodies at room temperature for 30 minutes.
[0145] Isolation of RNAs from Zkscan8-Introduced Umbilical Cord-Derived Mesenchymal Stem Cells (Msc-Zk8)
[0146] In order to investigate the expression of various genes upon introduction of Zkscan8, total RNAs were extracted using a HiYield total RNA mini kit (Real Biotech Corporation, Taiwan). After measuring absorbance at 260 nm and 280 nm using a spectrophotometer (NanoDrop, DE, USA), the total RNAs were quantified. Then, cDNAs were synthesized from 1 μg of the total RNAs using a Superscript II reverse transcriptase (Invitrogen, CA. USA).
[0147] Reverse Transcription Polymerase Chain Reaction (RT-PCR)
[0148] Maxime™ PCR PreMix (i-StarTaq, iNtRON, Sungnam, Korea) was used to conduct reverse transcription polymerase chain reaction (RT-PCR). 16 μL of RNase-free distilled water, 2 μL of cDNAs and 1 μL of primers per each were added and the reaction was conducted under the following conditions. Pre-denaturation was performed at 95° C. for 30 minutes, denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 1 minute. After repeating this procedure for a total of 32 times, followed by cooling at 72° C. for 5 minutes, the bands of the obtained PCR product were observed on 1% agarose gel.
TABLE-US-00001 TABLE 1 Target genes Primer sequences (5′ .fwdarw. 3′) Product length (bp) Zkscan8 SEQ ID NO 4 CGGAGGAAAGTCGGAAACCA 198 Zkscan8 SEQ ID NO 5 CTTCCCGTGGACCAAGAGTC 198 PPARγ2 SEQ ID NO 6 TTGGTGACTTTATGGAGCCC 311 PPARγ2 SEQ ID NO 7 CATGTCTGTCTCCGTCTTCT 311 aP2 SEQ ID NO 8 AAGAAGTAGGAGTGGGCTTT 285 aP2 SEQ ID NO 9 CCACCACCAGTTTATCATCC 285 Osteopontin SEQ ID NO 10 GAGACCCTTCCAAGTAAGTC 354 Osteopontin SEQ ID NO 11 GATGTCCTCGTCTGTAGCAT 354 ALP SEQ ID NO 12 TGGAGCTTCAGAAGCTCAAC 454 ALP SEQ ID NO 13 ATCTCGTTGTCTGAGTACCA 454 Sox6 SEQ ID NO 14 AACATGTGGCCTCCCATCTG 300 Sox6 SEQ ID NO 15 TCAGTGTGTCCACCACATCG 300 Aggrecan SEQ ID NO 16 TCAGGAGGGCTGGAACAAGT 350 Aggrecan SEQ ID NO 17 GGAGGTGGTAATTGCAGGGA 350 Mkx SEQ ID NO 18 GGCCACGAACACTACCATGA 175 Mkx SEQ ID NO 19 AGCTGCGCTTTCACCCTTAT 175 Egr-1 SEQ ID NO 20 CCAGTGGAGTCCTGTGATCG 206 Egr-1 SEQ ID NO 21 TCGCTCCTGGCAAACTTTCT 206 Egr-2 SEQ ID NO 22 TCTTCCTCTCTGGCCTACCC 400 Egr-2 SEQ ID NO 23 GTCTGTTGGGGTACTTGCGA 400 COL1a1 SEQ ID NO 24 AGTGGTTTGGATGGTGCCAA 170 COL1a1 SEQ ID NO 25 GCACCATCATTTCCACGAGC 170 COL3a1 SEQ ID NO 26 CTTCTCTCCAGCCGAGCTT 191 COL3a1 SEQ ID NO 27 CCAGTGTGTTTCGTGCAACC 191 Desmin SEQ ID NO 28 GAGGAAATCCGGCACCTCAA 253 Desmin SEQ ID NO 29 CATCCCCGTGTCTCGATGGTC 253 NEFL SEQ ID NO 30 CTGGAAATCGAAGCATGCCG 363 NEFL SEQ ID NO 31 GCGGGTGGACATCAGATAGG 363 GAPDH SEQ ID NO 32 AAATCCCATCACCATCTTCCAG 313 GAPDH SEQ ID NO 33 CATGAGTCCTTCCACGATACC 313
[0149] Quantitative Reverse Transcription Polymerase Chain Reaction (Quantitative RT-PCR)
[0150] The expression of the following genes was quantified in real time by quantitative reverse transcription polymerase chain reaction (quantitative RT-PCR; qRT-PCR) using Go Taq™ probe qPCR and RT-qPCR systems (Promega, WI, USA), TaqMan™ Gene Expression Assays (Applied Biosystems, Foster City, Calif., USA) and LightCycler 480 (Roche Applied Science, Mannhein, Germany); Scx (Hs03054634_g1), Mkx (Hs00543190_m1), Egr-1 (Hs00152928_m1), Egr-2 (Hs00166165_m1), Thbs4 (Hs00170261_m1), GAPDH (Hs99999905_m1). The polymerase chain reaction was conducted as follows. After repeating 50 cycles of pre-denaturation at 95° C. for 10 minutes, denaturation at 95° C. for 15 seconds, annealing at 60° C. for 1 minute and extension at 72° C. for 4 seconds, the reaction mixture was cooled at 40° C. for 30 seconds. Melting curve analysis was performed using the 2-ΔCt calculation method, and the result of qRT-PCR was analyzed with reference to the expression of GAPDH [Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods. 2001, 25:402-408].
[0151] Isolation of Proteins and Western Blotting
[0152] A tendon cell lysate was obtained from tendon cells as follows. After adding a lysis buffer (PRO-PREPTM, iNtRON, Sungnam, Korea) to tendon cell pellets, they were disrupted by vigorously vortexing for 10 seconds for 10 or more times. The resulting solution was centrifuged at 4° C. and at 13,000 rpm for 20 minutes, and a tendon cell lysate was obtained by recovering the supernatant. The tendon cell lysate was subjected to BCA for quantification of protein contents. The tendon cell lysates (10 μg) of the same concentration were loaded onto 10% acrylamide gel and proteins were separated based on size by electrophoresis (SDS-PAGE). The separated proteins were sufficiently soaked with a 1× transfer buffer (20% methanol, 0.025 M Tris base and 0.19 M glycine) and then transferred to a PVDF membrane by flowing current at 100 V for 90 minutes. The PVDF membrane was blocked for 1 hour in a 1× TBS-T solution (10 mM Tris pH7.5, 100 mM NaCl, 0.1% Tween 20) supplemented with 5% skim milk. Then, after adding primary antibodies to the 1× TBS-T solution at a ratio of 1:1000, reaction was performed by stirring at 4° C. The used primary antibodies are as follows: Zkscan8 antibody (STJ26239, St John's Laboratory) and R-actin antibody (ab170325, Abcam). The PVDF membrane reacted with the primary antibodies were washed sufficiently with 1× TBS-T for 10 minutes for 3 times or more. Then, after adding secondary antibodies (diluted to 1:20,000) to the 1× TBS-T solution, reaction was performed for 1 hour by stirring at room temperature. The used secondary antibodies are as follows: goat anti-mouse IgG-HRP (SA001-500, GenDEPOT) and goat anti-rabbit IgG-HRP (7074S, Cell Signaling). After washing 3 times with a 1× TBS-T solution for 10 minutes and treating with ECL, the PVDF membrane was imaged with ImageQuant LAS4000 mini (GE Healthcare Life Sciences, Little Chalfont, UK).
[0153] Design of Animal Experiment
[0154] All animal care and experimental procedures were approved by and conducted in accordance with the institutional experimental animal research committee (IACUC_2019_0044). 88 male Sprague-Dawley rats (12-week-old, 340-360 g) were divided into four groups (1) normal group (Normal); 2) physiological saline group (Saline); 3) umbilical cord-derived mesenchymal stem cell group (MSC); 4) Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8)).
[0155] The rats were anesthetized using Zoletil (30 mg/kg) and Rompun (10 mg/kg), and only the left shoulders of the rats were used in all the experiments. Surgery was conducted after slightly pressing the rat's sole with a fingernail to check if the anesthesia was successful. Then, the skin in front of the acromion of the left shoulder was incised by 2 cm. After exposing the supraspinatus tendon by incising the trapezius muscle and deltoid muscle attached to the acromion, a round full-thickness rupture tendon injury was made at a distance of 1 mm from the cartilage region of the supraspinatus tendon and the humeral head using a biopsy punch (BP-20F, Kai Medical Europe GmbH, Bremen, Germany) with a diameter of 2 mm (about 50% or larger of the tendon width). Then, 2) 10 μL of physiological saline, 3) umbilical cord-derived mesenchymal stem cells (1×10.sup.6 cells/10 μL physiological saline) or 4) Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (1×10.sup.6 cells/10 μL physiological saline) was injected to both sides of the ruptured tendon in two divided dosages using a 30-G needle. After suturing the trapezius and deltoid muscles with a 4-0 Vicryl suture (W9074, Ethicon, Cincinnati, Ohio, USA) and suturing the skin with Black Silk (SK439, AILee, Busan, Korea), the wound site was disinfected. After the surgery, the rats were allowed free cage activity.
[0156] The rats were sacrificed at weeks 2 and 4 after the surgery. The supraspinatus tendon of the rats was harvested and used for macroscopic, histological and biomechanical evaluation.
[0157] Macroscopic Evaluation
[0158] At 2 and 4 weeks after the surgery, the rats were sacrificed in a carbon dioxide chamber. The supraspinatus tendon of the rats was harvested along with the humeral head without removing the supraspinatus muscle. For macroscopic evaluation of tendon regeneration, Stoll's modified semi-quantitative evaluation system was used [Stoll C, John T, Conrad C et al. Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation. Biomaterials 2011; 32(21): 4806-4815]. The 12 parameters in the system were tendon rupture (breakage at the defective site), inflammation (swelling/redness or inflammation), tendon surface (tendon surface is not smooth but rough and uneven), neighboring tendon (the color, thickness and outer surface of the of tendon near the defective site turn abnormal), level of the defect (the defective site is filled and bulges as compared to the neighboring tendon), defect size (defect size is increased to 3 mm or larger), swelling/redness of tendon (the injured tendon turns red or swells), connection surrounding tissue and slidability (the injured tendon congeals with neighboring tissue without sliding (being separated) smoothly), tendon thickness (tendon thickness is increased as compared to the original thickness), color of tendon (bright white tendon has turn opaque and dark red), single strain of muscle (the supraspinatus tendon is intermixed with neighboring muscle and tissue, rather than only with the supraspinatus muscle), and transition of the construct to the surrounding healthy tissue (the connection between the defective site and neighboring healthy tendon is not smooth; the beginning of defect is clearly distinguished). Each parameter was given 0 or 1 point except for tendon swelling/redness (0 to 2) and tendon thickness (0 to 3). Therefore, the total macroscopic score varied between 0 (normal tendon) and 15 (most severe injury).
[0159] Histological Evaluation
[0160] After the macroscopic evaluation, the harvested tissues were immediately fixed in 4% (w/v) paraformaldehyde (PFA; Merck, Germany) for 24 hours, followed by decalcification in 10% EDTA (ethylendiaminetetraacetic acid, Sigma-Aldrich, St Louis, Mo., USA) for 2 days. After the decalcification, the tissues were dehydrated through an increasing series of ethanol gradient and defatted in chloroform. The fixed tissues were embedded in paraffin blocks and carefully trimmed to the appropriate middle site of tendon using a microtome, cut into 4-cm-thick sections and attached onto slides.
[0161] A randomly selected slide was stained with hematoxylin and eosin (H&E) and analyzed with an optical microscope (U-TVO 63XC; Olympus Corp., Japan). For the evaluation of tendinopathy, each slide was evaluated using Astrom and Movin's modified semi-quantitative grading scale [Jo C H, Shin W H, Park J W et al. Degree of tendon degeneration and stage of rotator cuff disease. Knee Surg Sport Tr A 2017; 25 (7): 2100-2108]. The 7 parameters of the system include: fiber structure (long collagen fibers are broken into small pieces), fiber arrangement (collagen fibers that have been arranged in parallel are arranged irregularly), rounding of the nuclei (the nuclei of fibroblasts that have been flat due to inactivation turn round as they are injured or activated), variations in cellularity (the number of cells in the tendon is increased and the cells form several populations), increased vascularity (the number and size of blood vessels in the tendon are increased significantly), decreased stainability (fibers constituting the tendon are decreased and stainability is decreased due to the decreased fiber density) and hyalinization (the tendon tissues consisting of collagen fibers turn glassy). The total degeneration score varied between 0 (normal tendon) and 21 (most severely degenerated).
[0162] For analysis of collagen fiber coherence (the state where the collagen fibers constituting tendon are coherently aligned in parallel), the slides were stained with picrosirius red and images were obtained using a circular polarization optical microscope. The collage fiber coherence, which is a measure of the extent of collagen fiber alignment in the major axis of the tendon, was quantified using a computer program called Orientation J plug-in for imageJ. Five regions were analyzed for each slide and the mean value multiplied by 100 was used as the final coherence value [Degen R M, Carbone A, Carballo C et al. The Effect of Purified Human Bone Marrow-Derived Mesenchymal Stem Cells on Rotator Cuff Tendon Healing in an Athymic Rat. Arthroscopy 2016; 32 (12): 2435-2443].
[0163] For evaluation of glycoaminoglycan (GAG)-rich area (formation of nonspecific cartilage tissue in tendon tissue) associated with heterotopic cartilage formation, the slides were stained with safranin O/fast green (Saf-O) and images were obtained using an optical microscope. The obtained images were analyzed with imageJ, and the glycoaminoglycan-rich area stained red was quantified.
[0164] The occurrence of heterotopic ossification (formation of nonspecific bone tissue in tendon tissue) was evaluated. It was evaluated that heterotopic ossification occurred when separated, clustered and bar-shaped foci were observed in the whole tendon structure on the slides stained with H&E. The area of heterotopic ossification was analyzed using imageJ.
[0165] Biomechanical Evaluation
[0166] At 2 and 4 weeks after the surgery, the rats were sacrificed in a carbon dioxide chamber. The tendon of the rats was harvested with the supraspinatus muscle attached to the humerus. Then, only the tendon was left by carefully removing the muscle. The harvested tissues were wrapped in saline-soaked gauze and kept at −80° C. Before testing, the tissues were thawed with saline-wetted gauze for 24 hours. The tissues were kept moist using saline-wetted gauze during all tests. The distal part of the humerus was vertically embedded in an aluminum tube full of polymethylmethacrylate (PMMA) in the custom-designed lower jig of a testing system. The proximal end of the tendon was fixed to the upper jig and compressed with sandpaper, gauge and rubber to prevent slippage and minimize damage of specimens. Testing was performed at 90° of abduction using a material testing system (H5K5, Tinus Olsen, England, UK) (
[0167] Statistical Analysis
[0168] All data were shown as mean±SD. The data were analyzed by one-way analysis of variance (ANOVA) and post-hoc analysis was performed using Bonferroni multiple comparison test. T-test was used to compare two groups. All statistical analyses were performed using the SPSS software version 23 (IBM). Differences of p<0.05 were considered statistically significant.
[0169] Experimental Results
[0170] Whole Transcriptome Profiling
[0171] For analysis of the expression patterns of tenogenesis, myogenesis, chondrogenesis and osteogenesis markers during embryonic stages, the expression patterns of a total of 25,070 expressed genes in embryonic stages E9.5-E15.5 were analyzed from whole transcriptome expression profiling datasets GSE30138, GSE54207 and GSE65180.
[0172] As seen from
[0173] Analysis of Gene Expression Pattern and PPI Network During Embryogenesis
[0174] Because analysis was conducted using three datasets, the similarity of expression patterns to marker genes was compared based on distance scores of the genes. 0 point was given if the expression pattern was identical to that of the marker genes in the three datasets. 0.33 point and 0.67 were given, respectively, if it is different in one and two datasets when the expression pattern began to increase. And, −0.33 point and −0.67 were given, respectively, if it is different in one and two datasets when the expression pattern began to decrease. Among the 9,163 DEGs, 149 genes with total distance scores of ±0.33 or lower, i.e., those showing difference in expression patterns in only one embryogenic stage, were screened primarily. The primarily screened candidate genes were subjected to PPI network, functional enrichment and module analyses while adjusting degree cut-off.
[0175] As a result of the protein-protein interaction (PPI) network analysis, three sub-clusters were identified. 99 genes were screened secondarily by excluding well-known genes. Then, 42 genes functionally related to tendon regeneration were screened tertiarily through gene ontology (GO). The tertiarily screened 42 genes were used for the final screening of genes related with tendon regeneration.
[0176] Comparison of Expression Pattern of Tenogenesis Markers and Zkscan8 Depending on Embryogenic Stages
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[0178] The expression pattern of the Zkscan8 gene was compared with that of tenogenesis markers depending on embryogenic stages to analyze their similarity. As shown in
[0179] Analysis of Zkscan8 Expression Pattern in Normal Tendon and Tendon with Rotator Cuff Disease
[0180] The expression patterns of tenogenesis markers and the Zkscan8 gene in the normal tendon obtained from a normal human and the tendon obtained from a patient with rotator cuff disease were investigated.
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[0182] As shown in
[0183] Characterization of Zkscan8-Introduced Umbilical Cord-Derived Mesenchymal Stem Cells (MSC-Zk8)
[0184] A pscAAV-GFP vector, which is a control group vector, and a pscAAV vector wherein Zkscan8 is inserted, are schematically shown in
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[0186] As shown in
[0187] As shown in
[0188] From the RT-PCR result, it can be seen that Zkscan8 was overexpressed in the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) and the control group (MSC-GFP) from 2 days after the introduction of the gene and the expression was decreased after 3 days.
[0189] From the western blotting result, it was confirmed that the expression of the Zkscan8 protein was increased in the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) by 1.5 times or higher as compared to the control group (MSC-GFP). Accordingly, it was confirmed that human umbilical cord-derived mesenchymal stem cells overexpressing the Zkscan8 protein were prepared successfully.
[0190] Evaluation of Differentiability of Zkscan8-Introduced Umbilical Cord-Derived Mesenchymal Stem Cells (MSC-Zk8) to Tendon
[0191] In order to investigate the relationship between the Zkscan8 gene and tendon differentiation, the umbilical cord-derived mesenchymal stem cells (UCMSCs) were cultured in a tendon differentiation medium containing connective tissue growth factor (CTGF) and ascorbic acid for 2 weeks to induce differentiation into tendon cells. The expression of Zkscan8 in the cells was investigated on days 3, 7 and 14. Then, after culturing the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) and the control group (MSC-GFP) for 3 days by 3D culture to induce differentiation into tendon, the expression of tendon-related genes (Scx, Egr-1, Egr-2, Thbs4) was confirmed by qRT-PCR and histological analysis.
[0192]
[0193] As shown in
[0194] At present, several transcription factors such as Scx, Mkx and Egr-1/2 are known to be involved in tendon development. In upper limbs, Scx and Sox9 are the first signals for tendon progenitor cell initiation, whereas Mkx and Egr-1/2 are the second signals for tendon differentiation and maturation. In particular, Egr-1/2 is known as an essential transcription factor for tendon differentiation. It has been found as a new DNA-binding protein involved in tendon differentiation in vertebrates by regulating the production of type 1 collagen. The expression pattern of tenogenesis-related transcription factors (scleraxis; Scx, mohawk; Mkx, early growth response-1/2; Egr-1/2) in the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) and the control group (MSC-GFP) over time was investigated by qRT-PCR.
[0195]
[0196] As shown in
[0197] The change in histological pattern and tendon differentiation markers of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) when the cells were 3D-cultured under uniaxial tension to induce differentiation into tendon and ligament was investigated.
[0198]
[0199] As shown in
[0200]
[0201] As shown in
[0202] Analysis of Expression Pattern of Fat, Bone, Cartilage, Tendon, Muscle and Nerve-Related Markers in Zkscan8-Introduced Umbilical Cord-Derived Mesenchymal Stem Cells (MSC-Zk8)
[0203] Tendon tissue has many similarities to bone, cartilage and muscle tissues. Therefore, it was investigated whether Zkscan8 also affects the markers of fat, bone, cartilage, tendon, muscle and nerve that constitute the musculoskeletal system, in addition to tendon tissue. Specifically, Zkscan8-introduced umbilical cord-derived mesenchymal stem cells (MSC-Zk8) were prepared and the expression pattern of various genes including musculoskeletal markers was analyzed 2 and 3 days later.
[0204]
[0205] Macroscopic Evaluation
[0206]
[0207]
[0208] As shown in
[0209] At week 4, the total macroscopic score of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) was 2.75±0.46. In contrast, the total macroscopic score was 9.00±0.00 (p<0.000) and 4.25±0.89 (p<0.000), respectively, for the physiological saline group and the umbilical cord-derived mesenchymal stem cell group, indicating remarkably sever injury as compared to MSC-Zk8. The MSC-Zk8 group showed less tendon swelling and redness as compared to other groups.
[0210] From these experiments, it can be seen that the overexpression of Zkscan8 significantly increases the ability of recovering from tissue injury the umbilical cord-derived mesenchymal stem cells.
[0211] When the rotator cuff is injured, adhesion occurs between the rotator cuff and surrounding tissues. This limits the motion of the patient and causes severe pain by aggravating the tendon injury. It can be seen that the pain and symptoms in a patient that may be caused by tendon injury can be prevented or treated in the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8).
[0212] Histological Evaluation
[0213]
[0214] As shown in
[0215] At week 4, the total degeneration score was 7.00±1.07 for the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8), and 16.25±0.89 (p<0.01) and 8.75±1.83 (p=0.05) for the physiological saline group (Saline) and the umbilical cord-derived mesenchymal stem cell group (MSC), respectively. The Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) showed significantly lower degeneration of tendon as compared to other groups.
[0216]
[0217] As shown in
[0218] At week 4 after the beginning of experiment, the collage fiber coherence score of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) was significantly than other groups as 47.76±5.77. Specifically, the collage fiber coherence score of the physiological saline group (Saline) was 19.86±2.97 (p<0.000).
[0219]
[0220] As shown in
[0221]
[0222] As shown in
[0223] From the above experimental results, it can be seen that the Zkscan8-introduced umbilical cord-derived mesenchymal stem cells can be used for clinical application in order to prevent or treat musculoskeletal diseases by promoting the regeneration of tendon tissues and helping the recovery of tendon tissues.
[0224] The treatment of the musculoskeletal system including tendon is unsuccessful mostly because the injured tendon is not recovered to normal tissue but replaced by scar tissue having irregular collagen fibers and many blood vessels. However, the treatment with the Zkscan8-overexpressed umbilical cord-derived mesenchymal stem cells resulted in increased alignment of collagen fibers as compared to when only the umbilical cord-derived mesenchymal stem cells were treated, so that the tendon at the defective site could be recovered to normal tendon tissue rather than being replaced by scar tissue. In addition, the clinical application of stem cells for treatment of the musculoskeletal system has been limited due to the increased risk of shoulder pain, re-rupture and complication as heterotopic cartilage formation and ossification are induced. However, the Zkscan8-overexpressed umbilical cord-derived mesenchymal stem cells suppressed heterotopic cartilage formation to a level comparable to that of the normal group. That is to say, Zkscan8 enables umbilical cord-derived mesenchymal stem cells to be clinically applicable by resolving the side effect. In addition, since it has the effect of preventing, alleviating and treating tendon tissue injury, it can be used for prevention and treatment of musculoskeletal diseases.
[0225] Biomechanical Evaluation
[0226]
[0227]
[0228]
[0229] As shown in
[0230] At 4 weeks after the surgery, the ultimate failure load of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) was 22.84±1.99. The ultimate failure load was 18.51±1.97 (p=0.016) for the physiological saline group (Saline) and 22.14±2.19 for the umbilical cord-derived mesenchymal stem cell group (MSC). That is to say, the tendon of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) showed significant enhancement and recovery as compared to the physiological saline group (Saline).
[0231]
[0232] As shown in
[0233] At 4 weeks after the surgery, the stiffness of the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) was 9.04±1.12. The stiffness was 7.22±0.62 (p=0.013) and 7.38±1.09 (p=0.029) for the physiological saline group (Saline) and the umbilical cord-derived mesenchymal stem cell group (MSC), respectively. That is to say, it can be seen that the stiffness was significantly increased in the Zkscan8-introduced umbilical cord-derived mesenchymal stem cell group (MSC-Zk8) as compared to the physiological saline group (Saline) or the umbilical cord-derived mesenchymal stem cell group (MSC).
[0234]
[0235]
[0236] As shown in
[0237] Tendon is the tissue which transmits the force of muscle to bone during the movement of the body. In the present disclosure, the “ultimate failure load” refers to the maximum load that the tendon can bear until it is broken, and the “stiffness” refers to the maximum force that the tendon can endure without deformation.
[0238] It was confirmed that the tendon treated with the Zkscan8-overexpressed umbilical cord-derived mesenchymal stem cells showed increase in ultimate failure load and stiffness as compared to the tendon treated only with the umbilical cord-derived stem cells. This result is consistent with the preceding results that the treatment of the Zkscan8-overexpressed umbilical cord-derived mesenchymal stem cells to injured tendon exhibits the effect of inhibiting inflammation of tendon tissue, preventing degenerative changes, recovering collagen fiber arrangement and inhibiting heterotopic cartilage formation. That is to say, when the Zkscan8-overexpressed umbilical cord-derived mesenchymal stem cells are applied to full-thickness rupture injury, they can regenerate the defective tissue of the injured tendon and improve tendon functions.