CN-121985969-A - Kit for reconstructing a cell-free biomedical device for regenerative medicine, biomedical device reconstructed thereby and related synthesis method

CN121985969ACN 121985969 ACN121985969 ACN 121985969ACN-121985969-A

Abstract

Kit for reconstitution of a cell-free biomedical device for regenerative medicine, characterized in that the kit comprises hyaluronic acid and heparin based biomaterials in lyophilized form, which are rehydrated by secreted groups containing growth factors and chemokines collected from cultured human Mesenchymal Stromal Cells (MSCs).

Inventors

Qinqia Kinnich
Pierre Giulio Konardi
Carlo Jero Fiorika
Jiao Wanna Pitaresi
Fabio Salvatore Palenbo

Assignees

李米德基金会
地中海器官移植和高级专科治疗研究所

Dates

Publication Date: 20260505
Application Date: 20240919
Priority Date: 20230921

Claims (13)

1. A kit for reconstitution of a cell-free biomedical device for regenerative medicine, characterized in that the kit comprises hyaluronic acid and heparin based lyophilized form of a biomaterial which is rehydrated by a secreted group containing growth factors and chemokines collected from cultured human Mesenchymal Stromal Cells (MSCs), which lyophilized biomaterial and the secreted group are frozen until the cell-free biomedical device is reconstituted, wherein the reconstituted biomedical device is ready for use.
2. Kit for the reconstitution of a cell-free biomedical device for regenerative medicine according to the preceding claim, characterized in that the lyophilized form of the biological material and the secretory group are sterilized.
3. Kit for the reconstitution of a cell-free biomedical device for regenerative medicine according to any preceding claim, characterized in that the weight content of heparin in the lyophilized biological material is adjusted to achieve a controlled release of the growth factors and chemokines present in the secretory group.
4. Kit for reconstitution of a cell-free biomedical device for regenerative medicine according to the preceding claim, characterized in that the heparin is comprised in the lyophilized biological material in a concentration range of 1 to 37.5 wt%, preferably in a concentration range of 1 to 21 wt%.
5. Kit for the reconstitution of a cell-free biomedical device for regenerative medicine according to any preceding claim, characterized in that the hyaluronic acid is present in the biological material in a concentration range of 62.5 to 99 wt%, preferably in the lyophilized biological material in a concentration range of 79 to 99 wt%.
6. A cell-free biomedical device ready to be used in regenerative medicine, characterized in that it is reconstituted with a kit according to any one of the preceding claims.
7. A method for synthesizing a cell-free biomedical device that is immediately available in regenerative medicine, the method comprising the steps of: -synthesizing an intermediate starting from at least one amino derivative of hyaluronic acid having an average molecular weight comprised in the range of 50kDa to 1000kDa, wherein the functionalization range in the amino groups is 25 mole% to 50 mole% relative to the hyaluronic acid repeat units; -crosslinking the amino derivative of at least one hyaluronic acid after it has been dispersed in an aqueous medium to obtain a hydrogel; -lyophilizing the hydrogel; -heparinizing the lyophilisate until a biomaterial based on heparin and the amino derivative of hyaluronic acid is obtained; -integrating the lyophilized biological material with a secreted group comprising growth factors and chemokines collected from cultured human Mesenchymal Stromal Cells (MSCs).
8. Method for synthesizing a cell-free biomedical device ready to use in regenerative medicine according to claim 7, characterized in that it provides a further step of sterilizing the lyophilized biological material between the step of heparinizing the lyophilisate and the step of integrating the biological material.
9. A method for synthesizing a cell-free biomedical device that is immediately available in regenerative medicine, the method comprising the steps of: -synthesizing an intermediate starting from at least one amino derivative of hyaluronic acid having an average molecular weight comprised between 50kDa and 1000kDa, wherein the functionalization range in amino groups is 25 mol% to 50 mol% relative to the hyaluronic acid repeating units; -crosslinking the at least one amino derivative of hyaluronic acid in the presence of heparin after its dispersion in an aqueous medium to obtain a heparinized hydrogel; -lyophilizing said heparinized hydrogel until a biomaterial based on heparin and an amino derivative of said hyaluronic acid is obtained; -integrating the lyophilized biological material with a secreted group comprising growth factors and chemokines collected from cultured human Mesenchymal Stromal Cells (MSCs).
10. Method for synthesizing a cell-free biomedical device ready to use in regenerative medicine according to claim 9, characterized in that between said step of lyophilizing said hydrogel and said step of integrating said biomaterial, a step of sterilizing said lyophilized biomaterial is provided.
11. The method for synthesizing a cell-free biomedical device ready to use in regenerative medicine according to claim 7, wherein said heparinizing step is performed by adjusting the weight content of heparin added to said intermediate product to achieve a controlled release of said growth factors and said chemokines present in said secretory group.
12. The method for synthesizing a cell-free biomedical device ready to use in regenerative medicine according to claim 8, characterized in that said crosslinking step is performed by adjusting the weight content of heparin added to said intermediate product to achieve a controlled release of said growth factors and said chemokines present in said secretory group.
13. The method for synthesizing a cell-free biomedical device ready-to-use in regenerative medicine according to claim 7, wherein said heparinizing step comprises a step of lyophilizing said heparinized biomaterial prior to said integrating step.

Description

Kit for reconstructing a cell-free biomedical device for regenerative medicine, biomedical device reconstructed thereby and related synthesis method Technical Field The present invention relates to a kit for reconstructing a cell-free biomedical device for regenerative medicine, to a cell-free biomedical device for regenerative medicine, and to a method for synthesizing a cell-free biomedical device for regenerative medicine. Background For about ten years we have heard much information about cell-free therapies for repairing damaged organs and tissues based on the secretory group. By secreted group is meant a group of soluble factors secreted by a cell. In particular, the secretory group of Mesenchymal Stromal Cells (MSC) is considered as an enhancer of regenerative medicine, is a mixture of bioactive factors capable of stimulating endogenous repair processes (MESENCHYMAL STEM CELLS: time to CHANGE THE NAME | -STEM CELLS Translational Medicine, volume 6, stage 6, month 6 of 2017, pages 1445-1451, ;Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine" - Int. J. Mol. Sci. 2017, 18(9), 1852）.) is considered safer than conventional cell transplantation based on the secretory group approach because it would limit the potential risks associated with cell administration (tumorigenicity, infection, immune response). Advantages of cell-free therapies include greater versatility of the secretory group, which can be mass produced, stored and divided into immediately available batches （"Stem cells as drug delivery methods: Application of stem cell secretome for regeneration " - Advanced Drug Delivery Reviews, volumes 82-83, month 2015, pages 1-11 in advance. Currently, secretome-based therapies are in the experimental phase and include many registered clinical trials. The key aspects of the therapy based on the secretory group relate to the stability and half-life of the bioactive factor contained therein, 1 month in （"Stem cell secretome, regeneration, and clinical translation: a narrative review" - Ann Transl Med.2021 years; 9 (1): 70), the therapeutic efficacy of the secretory group itself being dependent on the stability and half-life of the bioactive factor. Furthermore, since it is a soluble therapeutic agent, its administration requires some precautions that determine the way in which the soluble factor is "presented" to the damaged tissue, thereby affecting the therapeutic effect ("THE STEM CELL secretome and its role in brain repair" -Biochimie, volume 95, 12, month 12, 2013, pages 2271-22855). For example, there are support or release systems that ensure prolonged or controlled release of the secretory group by increasing the contact time with the damaged tissue/organ. At the same time, the delivery system ensures "stability" of the secretory group by providing protection from clearance and enzymatic degradation in vivo (Polymers for Drug DELIVERY SYSTEMS-annu Review of CHEMICAL AND Biomolecular Engineering, volume 1:149-173 (8 months 2010; hydrogels for protein DELIVERY IN tissue engineering-J Control Release, 7 months 20; 161 (2). 680-92). Known delivery systems include hydrogels, which are three-dimensional hydrophilic materials with high affinity for water, but which are insoluble due to the presence of intermolecular bonds that affect their physical and chemical properties, （Growth factor delivery from hydrogel particle aggregates to promote tubular regeneration after acute kidney injury - J Control Release 2013, 5, 10, ;167(3):248-55;Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds – J Control Release – 2015, 12, 28; 220 (PtA): 79-88). Hydrogels have wide applications in regenerative medicine and drug delivery due to their biocompatibility and swelling capacity. Generally, hydrogels based on natural polymers are preferred over hydrogels based on synthetic materials and are currently used for the treatment of skin wounds (wound healing) (Functional Hydrogels as Wound Dressing to Enhance Wound Healing-ACS Nano.2021, month 8, 24, ; 15(8):12687-12722;Local injection of high-molecular hyaluronan promotes wound healing in old rats by increasing angiogenesis - Oncotarget.2018; 9:8241-8252） and for cartilage regeneration （In situ forming hydrogels of new amino hyaluronic acid/benzoyl-cysteine derivatives as potential scaffolds for cartilage regeneration - Soft Matter Issue 18, 2012; In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration - Carbohydr Polym.2015, month 5, 20; 122:408-16). Natural polymers include glycosaminoglycans (GAGs), which are known to help regulate the biological functions of the extracellular matrix (ECM), including the physical and mechanical regulation of connective tissue and cellular activity. GAGs interact with growth factors and chemokines in the tissue microenvironment through specific epitopes, controlling their accumulation and diffusion within the cell and maintaini