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WO2025141613A1 - Matrice composite à base de fibroïne de soie utilisée en tant qu'échafaudage pour tissu - Google Patents

Matrice composite à base de fibroïne de soie utilisée en tant qu'échafaudage pour tissu Download PDF

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Publication number
WO2025141613A1
WO2025141613A1 PCT/IN2024/052439 IN2024052439W WO2025141613A1 WO 2025141613 A1 WO2025141613 A1 WO 2025141613A1 IN 2024052439 W IN2024052439 W IN 2024052439W WO 2025141613 A1 WO2025141613 A1 WO 2025141613A1
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WO
WIPO (PCT)
Prior art keywords
silk fibroin
composite
woven mesh
w2xc
woven
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Pending
Application number
PCT/IN2024/052439
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English (en)
Inventor
Anuya Amol NISAL
Swati Govind SHUKLA
Raeesa SAYYAD
Rucha Vikram DESHPANDE
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Serigen Mediproducts Private Ltd
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Serigen Mediproducts Private Ltd
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Publication of WO2025141613A1 publication Critical patent/WO2025141613A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments

Definitions

  • Tissue engineering involves use of scaffolds for regeneration and/or repair of damaged tissue. These meshes / matrices play a crucial role in tissue regeneration by providing a scaffold for cells to grow, proliferate and function. These matrices should have appropriate mechanical properties sufficient to withstand the stresses experienced by the native tissue. During surgical procedures, it is common practice to suture these scaffolds to nearby tissues. Therefore, it is important for these matrices to possess suturability and conformability, allowing them to be securely sutured and adapt to the surrounding tissue. In addition, it is also desirable that the matrices can be cut to the desired shape and size during the surgical procedure.
  • Acellular dermal matrix (ADM) and collagen matrices are currently used for soft tissue regeneration. They are easy to use, and do not require two surgeries. But use of ADMs or collagen matrices impose the high risk of infection transmission and complications like seroma, skin necrosis, haematoma, poor angiogenesis, poor tissue integration and implant loss. Other synthetic meshes are also explored for soft tissue regeneration application. Owing to the woven/knitted thread structure, they do not offer sufficient surface area for cell attachment and tissue integration resulting in complications such as capsular contracture, seroma, implant loss etc. Thus, there is indeed a need for development of a matrix that has desired mechanical properties, appropriate surface textures, possess suturability, conformability and optimal porosity. The matrix should also support cellular adhesion, proliferation, collagen deposition and new blood vessel formation (angiogenesis), ultimately resulting in soft tissue regeneration.
  • angiogenesis new blood vessel formation
  • Silk fibroin is a natural biopolymer extracted from silkworm Bombyx mori.
  • SF is an attractive biomaterial for tissue engineering due to biocompatibility, ease of processability, tuneable mechanical properties and resorption rates (Vepari et al., 2007; Nguyen et al., 2019; Sun W et al., 2021).
  • SF can be processed into various forms to support hard and soft tissue regeneration to achieve different porosities, mechanical properties and biological properties.
  • Figure 2 Depicts the percentage cellular adhesion at 24h for different composites.
  • Figure 7 Depicts the woven mesh of varying dimensions.
  • Bombyx mori commonly known as the domestic silk moth
  • the domestic silk moth was domesticated from the wild silk moth Bombyx mandarina, which has a range from northern India to northern China, Korea, Japan, and the far eastern regions of Russia.
  • the domestic silk moth derives from Chinese rather than Japanese or Korean stock.
  • the present invention discloses a three-dimensional biocompatible silk fibroin composite matrix for tissue regeneration comprising of one or more layers of i. Lyophilized regenerated silk fibroin (LR); and/or ii. Non-woven mesh of said silk fibroin; and/or iii. Woven mesh of said silk fibroin.
  • LR Lyophilized regenerated silk fibroin
  • the silk fibroin composites of the present invention comprise of; i. Lyophilized regenerated silk fibroin (LR) of thickness 1 to 3 mm, grammage of 5-35 mg/cm 2 and porosity of > 90-98%; and/or ii. One or more non-woven mesh/es (NW) with thickness of 0.2 to 3.0mm each, weight of 5-25mg/cm 2 and porosity of 70-90%; and/or iii.
  • LR Lyophilized regenerated silk fibroin
  • NW non-woven mesh/es
  • One or more woven mesh/es each with a pitch ranging between 1mm- 10mm in XY direction and/or layer/s woven at angles ranging from 30 to 60 deg to X and Y direction (XY-CC), thickness of 0.2 to 3.0 mm; grammage of 1-10 mg/cm 2 and porosity of 80 - 99%.
  • the silk fibroin composites of the present invention comprises; i. Lyophilized regenerated silk fibroin (LR) of thickness 1 to 3 mm, grammage of 5-35 mg/cm 2 and porosity of > 90-98%; and/or ii. Combination of Woven mesh and silk solution (W2XC-R) wherein the woven mesh has a pitch ranging between 1mm- 10mm in XY direction as well as one layer each woven at various angles ranging from 30 to 60 deg to X and Y direction (XY-CC) with the thickness of 1.0 to 3.0mm; weight of 9-12mg/cm 2 and porosity of 85-95%; iii.
  • Non-woven mesh and silk solution (NW+R) with the thickness of 1.0 - 3.0mm; weight of 18-24mg/cm 2 and porosity of 85-95%; iv. Combination of one woven mesh sandwiched between two non-woven mesh and silk solution (NW-W2XC-NW-R) of thickness 1.0 to 3.0mm, weight of 25-35mg/cm 2 and porosity of 75-80%; v. Combination of one woven mesh, one non-woven mesh and silk solution (W2XC-NW-R) with a thickness of 1.0 to 3.0mm, grammage of 15- 22mg/cm 2 and porosity of 85-95%.
  • the present invention discloses a method for preparing the silk fibroin composites comprising; i. Extracting the pure silk fibroin by boiling the Bombyx mori pure bivoltine silk hanks in alkaline solution until removal of sericin and obtaining the pure fibroin fibers; ii. Vacuum drying the pure fibroin fibers of step (i); iii. Dissolving the dried pure silk fibroin in LiBr followed by dialysis to obtain the silk fibroin solution with 4wt% - 6wt% concentration; iv. Diluting the solution of step (iii) with DI water to obtain 0.1wt% to 6wt% of silk fibroin (SF); v. Converting said silk fibroin (SF) solution of step (iv) to lyophilized SF and using it as is and/or with woven mesh/es and /or with non-woven mesh/es to form a composite as desired.
  • the process for preparing the Eyophilized Regenerated Silk Fibroin comprising; i. Freezing the silk fibroin solution (SF) as prepared above in suitable concentration in the mold at a temperature in the range of -10°C to -80°C for 1-24 hours; and ii. Lyophilizing at -55°C to -80°C for 5-24 hours to obtain the desired product followed by annealing and sterilization before use.
  • SF silk fibroin solution
  • Lyophilizing at -55°C to -80°C for 5-24 hours to obtain the desired product followed by annealing and sterilization before use.
  • the process for preparing the non-woven silk mesh comprising; i. Degumming the silk thread to obtain pure silk fibroin fibers as described above and soaking the pure silk fibroin fibers in distilled water for about 4-24 hours and beating to obtain the pulp; ii. Preparing the non-woven mesh from said pulp using hand sheet former machine and drying followed by annealing and sterilization before use.
  • the process for preparing the woven silk meshes comprising; i. Weaving the continuous monopoly/ multiply silk thread using a pitch (distance between the two silk threads) in the range of 1mm to 10mm to obtain mesh consisting of single layer woven in XY direction and single layer woven in criss-cross direction (at angles ranging from 30 to 60 deg to XY); ii. Applying the silk fibroin solution (SF) as prepared above in suitable concentration on to the woven mesh for sticking the threads together and drying to obtain the desired product followed by annealing and sterilizing before use.
  • SF silk fibroin solution
  • the process for preparing the W2XC-R (S4) composite comprising; i. Placing the woven silk fibroin mesh (W2XC) in a mold and pouring the silk fibroin solution as prepared above in appropriate concentration on to said woven mesh; ii. Freezing the composite at a temperature ranging between -10°C to -80°C for 1-24 hours and further freeze drying at -55°C to -80°C for 5-24 hours and sterilizing the W2XC-R composite before use.
  • W2XC woven silk fibroin mesh
  • the process for preparing the NW+R (S5) composite comprising; i. Preparing the non-woven mesh (NW) and silk fibroin (SF) solution as described above; ii. Placing the silk fibroin non-woven mesh in a mold and pouring the silk fibroin solution of appropriate concentration on to said non-woven mesh; iii. Freezing the composite at a temperature ranging between -10°C to -80°C for 1-12 hours and further freeze drying at -55°C to -80°C for 5-24 hours and steam sterilizing the NW+R composite before use.
  • NW non-woven mesh
  • SF silk fibroin
  • the process for preparing the NW-W2XC-NW-R (S6) composite comprising; i. Preparing two silk fibroin non-woven mesh (NW), woven mesh (W2XC) , and the silk fibroin (SF) solution as described above; ii. Placing the single woven mesh (W2XC) sandwiched between two nonwoven (NW) meshes in a mold and pouring the silk fibroin solution of appropriate concentration; iii.
  • Example 6 Evaluation of properties of the tissue scaffolding composite matrices
  • % cellular adhesion (Absorbance of Test at 590nm/ Absorbance of plate control at 590nm) *100
  • L929 cells were trypsinized and seeded on all matrices and an empty well without any sample (plate control), at a seeding density of 10,000 cells/well. Cells were then cultured in DMEM with 10% FBS and incubated at 37°C, 5% CO2 for 1, 4 and 7 days with media change at every 48h. Proliferation of cells cultured on different matrices was assessed by MTT assay.
  • MTT assay media was removed from each well. Matrices were washed with IX PBS two times. 150 pl of 0.5mg/ml MTT was added to each well. Cells were incubated with MTT for 4h at 37°C. After incubation, the MTT solution was discarded and the crystals were dissolved in 150pl DMSO. Absorbance was measured at 570nm. Data expressed as average absorbance at 570nm ⁇ SD.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RNA was measured using Nano-drop (ND-1000, UV / Vis spectrophotometer, Nano-drop technologies, USA).
  • cDNA was synthesized from 200ng of total RNA using Verso cDNA synthesis kit according to manufacturer’s instructions.
  • the Ang-1 represents the marker for blood vessel formation and Collagen 1 was used as collagen deposition marker.
  • P-actin was used as housekeeping gene control. Primers specific for these markers were used for RT- PCR.
  • PCR (Eppendorf Master cycler, realplex 2, ep gradient S) conditions used for 30 cycles of amplification were as follows:
  • PCR products were resolved on 1.2% agarose gel and visualized using SYBR gold nucleic acid gel stain (Invitrogen) on Bio-Rad, Molecular Imager, ChemiDoxTM XRS+ imaging system.
  • P-actin was used as housekeeping gene control. Bands were observed and documented using Gel documentation system. Band intensity was then analysed using Image J software. Values were normalized using P-actin (housekeeping gene control).
  • Silk fibroin composite displayed at least 1.5-fold increase in blood vessel formation and collagen deposition indicative of better tissue regeneration ability (Fig 4 and Fig 5).
  • Silk composite matrices were cut to with dimensions 2cm (length) X 1cm (width). Suture thread was passed through centre and knot was tied to the silk fibroin composite. The samples were clamped at the edge located opposite to the suture. Suture loop was pulled at a rate 0.05mm/s. Maximum load taken was calculated and expressed in newton (N) (ISO7198).
  • Silk fibroin composite displayed comparable suturability to that of collagen matrix. Suturability of the silk fibroin composite matrix annealed using organic solvent and sterilized using ethylene oxide was comparable with ADM and displayed 1.5-fold increase in suture retention ability (Fig 6).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Composite Materials (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La divulgation concerne des matrices composites à base de fibroïne de soie utilisées en tant qu'échafaudages dans la régénération tissulaire et leur procédé de préparation.
PCT/IN2024/052439 2023-12-28 2024-12-27 Matrice composite à base de fibroïne de soie utilisée en tant qu'échafaudage pour tissu Pending WO2025141613A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202321089314 2023-12-28
IN202321089314 2023-12-28

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WO2025141613A1 true WO2025141613A1 (fr) 2025-07-03

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2869857A1 (fr) * 2012-07-09 2015-05-13 Trustees Of Tufts College Fibroïne de soie de poids moléculaire élevé et ses utilisations
US20180272030A1 (en) * 2016-10-31 2018-09-27 Sofregen Medical, Inc. Compositions comprising low molecular weight silk fibroin fragments and plasticizers
WO2019094700A1 (fr) * 2017-11-10 2019-05-16 Cocoon Biotech Inc. Produits à base de soie et procédés d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2869857A1 (fr) * 2012-07-09 2015-05-13 Trustees Of Tufts College Fibroïne de soie de poids moléculaire élevé et ses utilisations
US20180272030A1 (en) * 2016-10-31 2018-09-27 Sofregen Medical, Inc. Compositions comprising low molecular weight silk fibroin fragments and plasticizers
WO2019094700A1 (fr) * 2017-11-10 2019-05-16 Cocoon Biotech Inc. Produits à base de soie et procédés d'utilisation

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