WO2023134090A1 - Utilisation d'un neuropeptide dans le traitement de maladie à lésion osseuse - Google Patents

Utilisation d'un neuropeptide dans le traitement de maladie à lésion osseuse Download PDF

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WO2023134090A1
WO2023134090A1 PCT/CN2022/091362 CN2022091362W WO2023134090A1 WO 2023134090 A1 WO2023134090 A1 WO 2023134090A1 CN 2022091362 W CN2022091362 W CN 2022091362W WO 2023134090 A1 WO2023134090 A1 WO 2023134090A1
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bone
neuropeptide
npvf
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scaffold
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Chao Zhong
Yanyi WANG
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Shenzhen Institute Of Advanced Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure generally relates to a method for enhancing angiogenesis and osteogenesis using a neuropeptide and a neuropeptide-containing fusion protein as self-assembling coatings that is mainly aimed for bone void filling procedures and repair, regeneration or formation of cartilage, bone or a combination thereof in the subject.
  • NPVF is one of those critical neuropeptides, which belongs to NPFF family.
  • the NPFF system belongs to the RF-amide neuropeptide family. It was first identified from bovine brain extract through isolation of the endogenous peptides that had cross immunoreactivity with cardio-excitatory peptide FMRF-NH2 (Yang, H.Y., et al., Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci U S A, 1985. 82 (22) : p. 7757-61.; Mouledous, L., C. Mollereau, and J.M.
  • Kanaya, and H. Ozawa Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization. Histochemistry and cell biology, 2021. 155 (4) : p. 465-475.; Lin, Y., et al., NPFFR2 Activates the HPA Axis and Induces Anxiogenic Effects in Rodents. International journal of molecular sciences, 2017. 18 (8) . ) . Those functions include food intake, blood pressure, memory, insulin release, neural regeneration, and cardiovascular activity (Panula, P., A. Aarnisalo, and K. Wasowicz, Neuropeptide FF, a mammalian neuropeptide with multiple functions.
  • the present disclosure provides use of a neuropeptide or a pharmaceutical composition comprising a neuropeptide in the preparation of product for inducing or enhancing repair, regeneration or formation of dental, cartilage, bone or a combination thereof in the subject.
  • the product comprises medication, kit, or medical equipment.
  • the present disclosure provides a method for inducing or enhancing repair, regeneration or formation of dental, cartilage, bone or a combination thereof in the subject comprising administering to the subject an effective amount of neuropeptide or pharmaceutical composition comprising a neuropeptide.
  • the present disclosure provides a neuropeptide or a pharmaceutical composition comprising a neuropeptide for inducing or enhancing repair, regeneration or formation of dental, cartilage, bone or a combination thereof in the subject.
  • the subject is afflicted with a dental, cartilage or bone defect, fracture, disorder or disease.
  • the subject is in need of bone void filling in the procedure of dental implant, periodontics, maxilla-craniofacial orthopedics.
  • the cartilage defect, fracture, disorder or disease comprises a full or partial thickness articular cartilage defect; osteochondral defect; osteoarthritis; a joint defect; or a defect resulting from trauma, sports, or repetitive stress.
  • the bone defect, fracture, disorder or disease comprises bone defect, bone fracture, osteoporosis, osteomalacia, metabolic bone diseases, trauma-caused bone fracture, stress fracture, bone cancer, and joint diseases containing osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, lupus, gout or bursitis.
  • the pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, the routes of administration include local injection.
  • the pharmaceutical composition of the invention administered via a device, e.g., stent or catheter, impregnated or coated with the active neuropeptide or recombinant protein.
  • the present disclosure provides use of a neuropeptide or a pharmaceutical composition comprising a neuropeptide in the preparation of medication for treating ischemia vascular disease in the subject.
  • the present disclosure provides a method for treating ischemia vascular disease in the subject with an effective amount of neuropeptide or pharmaceutical composition comprising a neuropeptide.
  • the present disclosure provides a neuropeptide or a pharmaceutical composition comprising a neuropeptide for treating ischemia vascular disease in the subject.
  • the present disclosure provides use of a neuropeptide or a pharmaceutical composition comprising a neuropeptide in the preparation of medication for enhancing osteogenesis and/or angiogenesis in the subject.
  • the present disclosure provides a method for enhancing osteogenesis and/or angiogenesis in the subject with an effective amount of neuropeptide or pharmaceutical composition comprising a neuropeptide.
  • the present disclosure provides a neuropeptide or a pharmaceutical composition comprising a neuropeptide for enhancing osteogenesis and/or angiogenesis in the subject.
  • the subject is a human subject or an animal subject.
  • the neuropeptide comprises or is a RFamide which is selected from the group consisting of PrRP family, NPFF family, RFRP family, KiSS-1 family, and QRFP family, preferably, the NPFF family comprises or is selected from the group consisting of NPFF, NPAF, NPSF, RFFP-1, and NPVF (RFFP-3) , more preferably, the neuropeptide comprises or is NPVF.
  • the neuropeptide is endogenous and derived from mammalian, preferably, the mammalian comprises human, Rat, Mouse, and Bovine.
  • the NPFF comprises SQAFLFQPQRF (Human, SEQ ID NO: 5) , SPAFLFQPQRF (Bovine or Mouse, SEQ ID NO: 6) , or NPAFLFQPQRF (Rat, SEQ ID NO: 7) ;
  • the NPAF comprises AGEGLNSQFWSLAAPQRF (Human, SEQ ID NO: 8) , AGEGLSSPFWSLAAPQRF (Bovine, SEQ ID NO: 9) , QFWSLAAPQRF (Mouse, SEQ ID NO: 10) , or EFWSLAAPQRF (Rat, SEQ ID NO: 11) ;
  • the RFFP-1 comprises MPHSFANLPLRF (Human, SEQ ID NO: 12) , MPPSAANLPLRF (Bovine, SEQ ID NO: 13) , VPHSAANLPLRF (Mouse or Rat, SEQ ID NO: 14) ;
  • NPVF comprises VPNLPQRF (Human, S
  • the neuropeptide comprises an amino acid sequence of PQRF (SEQ ID NO: 1) or PLRF (SEQ ID NO: 4) at the terminal.
  • NPVF is a polypeptide comprising an amino acid sequence of X 1 PX 2 LPQRF (SEQ ID NO: 2) , X 1 is V or F, X 2 is N or S.
  • NPVF is a polypeptide comprising:
  • VPNLPQRF an amino acid sequence of VPNLPQRF (SEQ ID NO: 3) ;
  • the NPVF can enhance osteogenesis and/or angiogenesis in the subject.
  • the NPVF is a polypeptide with or without a tag
  • the tag is an affinity tag for protein purification such as staphylococcus protein A, glutathione-S-transferase, maltose-binding protein, cellulose-binding protein, chitin-binding domain, thioredoxin, strepavidin, RNaseI, polyhistidine, human growth hormone, ubiquitin, or antibody epitopes, solubility-enhancing tag such as Maltose binding protein, NusA, Glutathione-S-transferase, TrxA, Fh8, SUMO, Z-tag or Halo tag, or tags for reporter expression such as ⁇ -galactosidase, luciferase, or fluorescent proteins
  • the tag also can be selected from c-Myc, His, HA, GST, MBP, Flag, and Arg6.
  • the subject is non-human mammal or human.
  • the present disclosure provides a recombinant protein comprising a neuropeptide and an amyloid protein as self-assembling coatings.
  • the neuropeptide comprises a RFamide selected from the group consisting of PrRP family, NPFF family, RFRP family, KiSS-1 family, and QRFP family, preferably, the NPFF family is selected from the group consisting of NPFF, NPAF, NPSF, and NPVF, more preferably, the neuropeptide is NPVF;
  • amyloid protein is selected from the group consisting of CsgA, A ⁇ (1-42) , TTR1, ⁇ -synuclein, Sup35, Fus, TDP43, CP43, Ure2p, insulin ⁇ -Lactoglobulin, PTL, Mfp, hydrophobin, elastin, BSA.
  • the neuropeptide comprises an amino acid sequence of PQRF or PLRF at the terminal.
  • the NPVF is a polypeptide comprising an amino acid sequence of X1PX2LPQRF (SEQ ID NO: 2) , X1 is V or F, X2 is N or S, preferably, NPVF is a polypeptide comprising:
  • VPNLPQRF an amino acid sequence of VPNLPQRF (SEQ ID NO: 3) ;
  • the NPVF can enhance osteogenesis and/or angiogenesis in the subject, or can induce or enhance repair, regeneration or formation of cartilage, bone or a combination thereof in the subject.
  • NPVF comprises a polypeptide comprising an amino acid sequence of VPNLPQRF (SEQ ID NO: 3) and the protein as coatings is CsgA, preferably, the NPVF is conjugated to the N-terminus or C-terminal of an amyloid protein CsgA, more preferably, the NPVF is conjugated to the N-terminus or C-terminal of an amyloid protein CsgA by a linker.
  • the recombinant protein is with or without a tag, preferably, the tag is selected from c-Myc, His, HA, GST, MBP, Flag, and Arg6.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the neuropeptide of present disclosure or the recombinant protein of present discourse, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a scaffold coated with self-assembling recombinant protein of present discourse.
  • the scaffold is a 3-dimensional porous hydroxyapatite scaffold, bone implant, artificial bone graft, synthetic ceramic cement, ceramic granules and synthetic bone substitute material.
  • the scaffold approximates the form of a cylinder, cone, screw, rectangular bar, plate, disc, pyramid, granule, ball or cube.
  • the scaffold comprises a hollow or hollows along a Cartesian coordinate axis of said scaffold.
  • the scaffold further comprises a biocompatible polymer that is incorporated within at least a portion of said scaffold.
  • the scaffold is seeded with a cell population, which population comprises mesenchymal stem cells, osteoblasts, osteocytes, osteoclasts chondroblasts, chondrocytes, fibroblasts, or a combination thereof.
  • the present disclosure provides use of the recombinant protein of present discourse or the scaffold of present discourse in the preparation of product for inducing or enhancing repair, regeneration or formation of cartilage, bone or a combination thereof in the subject.
  • the product comprises medication, kit, or medical equipment.
  • the present disclosure provides a method for inducing or enhancing repair, regeneration or formation of cartilage, bone or a combination thereof in the subject with an effective amount of recombinant protein of present discourse or the scaffold of present discourse.
  • the subject is afflicted with a cartilage or bone defect, fracture, disorder or disease.
  • the cartilage defect, fracture, disorder or disease comprises a full or partial thickness articular cartilage defect; osteochondral defect; osteoarthritis; a joint defect; or a defect resulting from trauma, sports, or repetitive stress.
  • the bone defect, fracture, disorder or disease comprises bone defect, bone fracture, osteoarthrosis, or osteomalacia.
  • the subject is in need of bone void filling in the procedure of dental implant, periodontics, maxilla-craniofacial orthopedics.
  • the present disclosure provides use of the recombinant protein of present discourse or the scaffold of present discourse in the preparation of medication treating ischemia vascular disease in the subject.
  • the present disclosure provides a method for treating ischemia vascular disease in the subject with an effective amount of recombinant protein of present discourse or the scaffold of present discourse.
  • the present disclosure provides a recombinant protein of present discourse or the scaffold of present discourse for treating ischemia vascular disease in the subject.
  • the present disclosure provides use of the recombinant protein of present discourse or the scaffold of present discourse in the preparation of medication for osteogenesis and/or angiogenesis in the subject.
  • the subject is non-human mammal or human.
  • Figure 1 shows NPVF significantly promotes the migration and tube formation of HUVECs.
  • A The expression level of NPFFR1 in HUVECs.
  • B The proliferation of HUVECs treated with NPVF was evaluated by the CCK 8 assay.
  • C-D The wound healing of HUVECs.
  • E-F The Transwell migration of HUVECs.
  • G-I The tube formation of HUVECs.
  • J-L The gene expression level of PDGF, EGF and VEGF treated with NPVF on HUVECs were detected.
  • M The protein expression level of VEGF was investigated by NPVF on HUVECs.
  • Figure 2 shows preparation and characterization of the CsgA-NPVF nanofiber coating.
  • A Schematic diagram depicting the induced expression of recombinant protein and the preparation of nanofiber coating.
  • B Congo red staining of the purified CsgA and CsgA-NPVF nanofibers;
  • C Assembly kinetics of CsgA and CsgA-NPVF monomer solutions detected by ThT fluorescence assay;
  • D AFM images showing the morphology of the self-assembled protein nanofibers;
  • E Adsorption behavior comparison of CsgA and CsgA-NPVF nanofibers on HA substrates measured by QCM;
  • F SEM images showing the surface morphology of the 3D HA scaffold before and after the coating process.
  • Figure 3 shows CsgA-NPVF-coated scaffolds promotes the bone defect in rats.
  • A Micro-CT results of 3D reconstructed and 2D sagittal images of the bone defects.
  • B-C The histological analysis of the new bone formation by H&E and Masson.
  • E-G The histological analysis of CD31, VEGF and OCN staining.
  • Figure 4 shows cytocompatibility of the CsgA-NPVF nanofiber coating for cells.
  • A The SEM images of BMSCs on calcium phosphate scaffolds with or without the CsgA-NPVF nanofiber coating.
  • B The images of BMSCs adhering on calcium phosphate scaffolds with or without the CsgA-NPVF nanofiber coating stained with DAPI (nuclei, blue) and Rhodamine phalloidin (cytoskeleton, red) .
  • DAPI nuclei, blue
  • Rhodamine phalloidin cytoskeleton, red
  • Figure 5 shows CsgA-NPVF scaffolds promotes the repair of bone defect in rats.
  • A-B The quantification of BMD and BV/TV in the new bone formation.
  • Figure 6 shows the histological analysis of NPFFR1 staining in rat calvaria.
  • a polypeptide means one polypeptide or more than one polypeptide.
  • Neuropeptide used herein refers to a polypeptide comprising or is RFamide, e.g. PrRP family, NPFF family, RFRP family, KiSS-1 family, and QRFP family.
  • the neuropeptide comprises an amino acid sequence of PQRF or PLRF at the terminal.
  • NPFF family comprises NPFF, NPAF, NPSF, or NPVF.
  • NPVF is a polypeptide comprising an amino acid sequence of X 1 PX 2 LPQRF (SEQ ID NO: 2) , X 1 is V or F, X 2 is N or S.
  • NPVF is a polypeptide comprising an amino acid sequence of VPNLPQRF (SEQ ID NO: 3) .
  • the neuropeptide can be endogenous and derived from mammalian, preferably, the mammalian comprises human, Rat, Mouse, and Bovine.
  • the neuropeptide can be artificial and comprises an amino acid sequence at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity to endogenous neuropeptide e.g. NPVF (SEQ ID NO: 2 or 3) , and comprises PQRF at the terminal; or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids compared with SEQ ID NO: 2 or 3.
  • the neuropeptide also comprises the derivatives.
  • derivatives refers to a truncated or its mutation and the polypeptide.
  • derivatives may be a truncated or an amino acid sequence an amino acid sequence with at least about 70%, about 80%, about 85%, about 90%, about 95%, about 99%, or more identity, or an amino acid sequence with addition, deletion and/or substitution of one or more amino acids.
  • the term “subject” includes any human or nonhuman animal.
  • nonhuman animal includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.
  • treat refers to reversing, ameliorating or inhibiting the progression of the disease to which the term is applied, or one or more symptoms of the disease.
  • the term also include prevention of disease, which includes the prevention of disease or the onset of any symptoms associated therewith, and ameliorating symptoms or reducing the severity of any condition before its onset.
  • “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be 20 incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include local injection, e.g., intravenous (e.g., a peripheral vein, such as found in the extremities) , intraperitoneal, intradermal, subcutaneous, subdermal, oral, intranasal, aerosol (e.g., inhalation) , transdermal (i.e., topical) , transmucosal, vaginal, intrauterine, and rectal (e.g., suppositories) administration.
  • intravenous e.g., a peripheral vein, such as found in the extremities
  • intraperitoneal intradermal
  • subcutaneous subcutaneous
  • subdermal oral
  • aerosol e.g., inhalation
  • transdermal i.e., topical
  • transmucosal i.e., vaginal, intrauterine
  • rectal e.g., suppositories
  • Injectable solutions containing active neuropeptide or recombinant protein of the present invention may be administered to the vascular lumen of vessels (e.g., aorta or jugular vein) , or locally administered to bone defect or bone diseases.
  • active neuropeptide or recombinant protein of the present invention may be administered via a device, e.g., stent or catheter, impregnated or coated with the active neuropeptide or recombinant protein.
  • the embryonic kidney 293T cells (HEK93T) and human BMSCs were purchased from the cell bank of the Chinese academy of sciences (Shanghai, China) , and cultured with Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) and ⁇ minimum essential medium ( ⁇ -MEM, Gibco) with 10%fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) , 1%penicillin/streptomycin (Invitrogen) at 37°C with 5%CO 2 , respectively.
  • FBS fetal bovine serum
  • Human umbilical vein endothelial cells were purchased from Procell (Wuhan, China) and cultured with endothelial cell medium (ECM, ScienCell, CA) .
  • the mRNA sequence of the BMSCs were performed by LC-bio (Hangzhou, China) .
  • Cell Counting Kit-8 (CCK-8, Beyotime, Jiangsu, China) was performed to detect the proliferation of BMSCs and HUVECs after NPVF treatment.
  • 96-well plate was seeded with 5 ⁇ 10 3 cells per well in 100 ⁇ L medium at day 0. At day 1, 3, 5 and 7, the medium was refreshed with 10 ⁇ L CCK-8 solution and 90 ⁇ L medium, then the plate was incubated in an incubator for 2h. Finally, the absorbance values of the supernatants of each well were detected at 450 nm and the medium was refreshed.
  • Wound healing assay was carried out by seeding with HUVECs at 5 ⁇ 10 5 per well on 6-well culture plate. Then, the confluent cells were scratched by a sterile pipette tip. Cells were then incubated by conditional medium and images of the wounds were captured at 0 h and 36 h. Image J was used to calculated the rates of wound healing.
  • Transwell assay was performed to test the migration of HUVECs after the treatment of NPVF, RF9 (Selleck, Shanghai, China) , miR-NC mimic, miR-181c-3p mimic, NC siRNA and AGO1 siRNA (Ruibo, Guangdong, China) .
  • HUVECs were incubated in 6-well plate. After 80%confluence, cells were incubated by serum free medium for 48 h for testing the expression level of VEGF. Then an ELISA kit (Neobioscience, Shenzhen, China) was used to detect the supernatant liquor for analysis of VEGF content. The absorbance values at 450 nm were calculated and performed to calculate the VEGF content at the basis of the standard curve.
  • NPVF NPVF
  • RF9 and JW74 JW74
  • conditional medium 2 ⁇ 10 5 cells were seeded in 6-well plates with ⁇ -MEM.
  • Osteogenic medium (Cyagen, Suzhou, China) was refreshed to induce osteogenic differentiation after 80%confluence every two days.
  • a LEICA microscope was used to captured images.
  • Quantitative real-time polymerase chain reaction QPCR
  • BMSCs or HUVECs were incubated in 6-well plates with conditional medium for 48 h, and then total mRNA was extracted on the basis of the manufacturer’s protocols (EZBioscience, USA) .
  • Reverse transcriptase reactions had the purified RNA and 50 nM RT primer.
  • QPCR was performed on an MX3005P system by a SYBR Premix Ex Taq protocol (EZBioscience) .
  • GAPDH was set as the internal gene of mRNA expression. The primers were listed in Table 1.
  • BMSCs or HUVECs were lysed by RIPA lysis buffer (1 mM PMSF, Beyotime) .
  • the BCA protein assay kit (Beyotime) was performed to test the protein concentrations according to the manufacturer’s protocols. Equal amount of protein (35 ⁇ g) was electrophoresed on 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) , then transferred to a PVDF membrane. Membrane was blocked with 5% (w/v) milk and incubated overnight at 4 °C with the primary antibodies against GAPDH, AGO1, ⁇ -catenin (CST, Shanghai, China) .
  • the PVDF membrane was washed and incubated with an HRP-conjugated second antibody. Finally, the membrane was washed and reacted with the ECL detection kit (Thermo Scientific, Waltham, MA) . A scanning densitometry was used to quantified the signals.
  • Plasmid containing AGO1 wild or mutant 3′-UTR was fused to the 3′end of psiCHECK2 luciferase vector (Promega; Madison, WI) .
  • the wild type and mutant fragments of the AGO1 3′-UTR including the predicted miR-181c-3p target sites (Positions 3932-3938) were directly synthesized (Obio, Shanghai, China) .
  • the Lipofectamine 3000 reagents (Life Technologies) was used to transfect plasmids into the HEK293T cells with miR-181c-3p mimic or miR-NC (Ruibo) .
  • a dual-luciferase reporter assay kit (Promega) was used to detect the activities of Renilla and firefly luciferases after 48 h transfection. The normalizing Renilla luciferase activity to the firefly luciferase activity was used to calculate the relative luciferase activities.
  • Nanofiber solutions at an initial concentration of 0.5 mg ⁇ ml -1 were introduced into HA coated QCM chips at a flow rate of 0.02 ml/min using a four-channel Ismatec ICP-N4 peristaltic pump, followed by washing with PBS buffer at the same rate.
  • modified 3D HA scaffolds were washed with copious deionized water and dried using vacuum drying oven, then coated with an 8–10-nm gold layer using a sputter coater for SEM observation.
  • SEM images were taken using a JSM 7800 SEM operated at a 5-kV accelerating voltage in secondary electron imaging mode.
  • the BMSCs co-incubated with scaffolds were stained by 4', 6-diamidino-2-phenylindole (DAPI, blue) /Rhodamine Phalloidin (red) to directly observe the subtle cytoskeletal change by a LEICA microscope.
  • DAPI 6-diamidino-2-phenylindole
  • Rhodamine Phalloidin red
  • the rat skulls were scanned by a micro-CT scanner (Skyscan, kontich, Belgium) . Subsequently, tomograms were reconstructed with the 3D creator software (Skyscan) and the CTAn image analysis software was performed to calculate the ratio of bone volume/tissue volume (BV/TV) and the bone mineral density (BMD, g ⁇ cm -3 ) according to the reconstructed images. Then, samples were decalcified, embedded and cut (5- ⁇ m) . subsequently, the sections were stained with hematoxylin & eosin (H&E) , Masson trichrome, immunohistochemical staining (NPFFR1, CD31, VEGF and OPN) . The images were captured by a LEICA microscope.
  • Example 1 NPVF induces HUVECs migration and angiogenesis
  • NPFF receptors The expression level of NPFF receptors in HUVECs by qPCR was investigated. The results showed that the Ct values of NPFFR1 in HUVECs were 19.20 ⁇ 0.22, indicating that NPFFR1 was highly expressed in HUVECs ( Figure. 1A) . Next, the effect of NPFFR1 on HUVECs through selective agonist NPVF was further explored. The CCK-8 assay indicated that low-dose NPVF (0.01, 0.1, and 1 nM) had a slight promotion effect on HUVECs proliferation. dose of NPVF (0.01, 0.1, and 1 nM) showed an increased effect on the proliferation of HUVECs (Figure. 1B) .
  • the NPVF group had more loop structures and a higher number of branch points compared with the control group, while RF9 reversed the promotion of HUVECs tube formation induced by NPVF.
  • qPCR and ELISA assay were employed to detect the expression of angiogenesis-associated genes in HUVECs.
  • the qPCR results revealed that VEGF, EGF, and PDGF gene expression in HUVECs remarkably increased after NPVF treatment, and this promotion effect could be offset by the addition of RF9.
  • Figure. 1J-1L ELISA analysis of the protein expression level of VEGF yielded the same conclusion.
  • Figure. 1M Taken together, the experimental results suggested that NPVF significantly promotes migration and angiogenesis via activating NPFFR1 in HUVECs.
  • Example 2 CsgA-NPVF-coated scaffolds enhanced bone regeneration in vivo
  • NPVF could induce angiogenesis of HUVECs.
  • angiogenesis is one of the major key events in the process of bone regeneration.
  • CsgA-NPVF a functional recombinant protein, by genetic fusion of NPVF at the N-terminus of an amyloid protein CsgA, the major protein component of E. coli biofilm that is well known for its universal coating formation ability towards a given substrate through amyloid self-assembly.
  • the CsgA-NPVF protein could form robust protein nanofiber coatings on the surface and the internal interfaces of a porous 3-D hydroxyapatite scaffold through overnight solution incubation (Figure 2A) .
  • This strategy offers a facile way to display functional peptides on the amyloid scaffolds and overcomes the instability and low bioavailability of peptides.

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Abstract

La présente invention concerne un procédé d'amélioration de l'angiogenèse et l'ostéogenèse à l'aide d'un neuropeptide de façon à traiter une maladie à lésion osseuse. La présente invention concerne également une protéine recombinante comprenant un neuropeptide et une protéine en tant que revêtements à auto-assemblage et l'utilisation d'une protéine recombinante pour le traitement d'une maladie à lésion osseuse.
PCT/CN2022/091362 2022-01-13 2022-05-07 Utilisation d'un neuropeptide dans le traitement de maladie à lésion osseuse WO2023134090A1 (fr)

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Citations (2)

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CA2223049A1 (fr) * 1995-06-07 1996-12-19 Stryker Corporation Produits osteogeniques sterilises en fin de fabrication et leur preparation
CN112972766A (zh) * 2021-02-22 2021-06-18 苏州大学 高机械强度丝素蛋白-羟基磷灰石复合骨支架及其制备方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2223049A1 (fr) * 1995-06-07 1996-12-19 Stryker Corporation Produits osteogeniques sterilises en fin de fabrication et leur preparation
CN112972766A (zh) * 2021-02-22 2021-06-18 苏州大学 高机械强度丝素蛋白-羟基磷灰石复合骨支架及其制备方法

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Title
GAO Y， ET AL.: "Effect of neuropeptides on periodontium and oral bone repair", CHINESE JOURNAL OF TISSUE ENGINEERING RESEARCH, vol. 18, no. 11, 12 March 2014 (2014-03-12), XP009547731 *
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