Patent Description:
Articular surfaces of the joints, such as knee or shoulder joint, are covered with cartilage tissue to prevent direct friction of the bones that make up the joint. A damage of the cartilage tissue due to aging, heavy or repetitive overloads lead to inflammation of the joint, which causes pain and movement disorders and gradual development of osteoathritis. In fact, articular cartilage tissue damage is often found in both young and elderly patients. In order to alleviate the symptoms and improve quality of patient's life there is growing demand for more efficient methods to restore damaged articular cartilage which has no intrinsic capacity to regenerate on its own. The biggest challenge in the treatment of damaged articular cartilage is to develop the optimal method of cartilage repair, capable of regenerating long lasting hyaline cartilage.

Due to the increased clinical need to restore articular cartilage, improved tissue repair techniques and new tissue engineering products continue to be developed. These products are mainly related to the use of biomaterials for the development of osteochondral implants to restore anatomically the bilayer structure of natural osteochondral tissue while maintaining mechanical properties of repair tissues. <CIT> relates to an adhesion preventing layer which can prevent abnormal tissue adhesion and, more specifically, to an adhesion preventing layer having a multi-layered nanofibrous structure manufactured by an electrospinning method and containing a hydrophilic natural polymer such as chitosan and the like and a manufacturing method thereof. The manufacturing method is comprised of: electrospinning a mixed polymer solution including a biodegradable natural polymer to form a hydrophilic substrate layer on non-woven fabric; and electrospinning a mixed polymer solution including a hydrophobic biodegradable synthetic polymer on top thereof to form a hydrophobic layer on the non-woven fabric, thereby allowing the hydrophobic layer to inhibit cell proliferation toward the wound portion in an organ adjacent to the abdominal wall while a wound is healed by the hydrophilic layer and keep playing a role of a physical barrier until the wound is healed.

<CIT> belongs to the field of a biomaterial, and more specifically relates to an oriented porous composite electrospinning fiber scaffold having a bionic surface. The oriented porous composite electrospinning fiber scaffold is formed by composite fibers with single oriented arrangement and having a surface distributed with nano pores. The composite fibers takes the L-polylactide electrospinning fiber with the single oriented arrangement and having the surface distributed with nano pores as a matrix. The surface of the matrix is modified with polydopamine, or the matrix surface modified with polydopamine is grafted with a cartilage restoration-promoting drug.

<CIT> provides a synthetic anisotropic, layered construct which exhibits zonal organisation and can be used to assist with cartilage formation. The invention also provides an electrospinning process for producing the construct. The construct mimics articular cartilage in terms of fibre organization and mechanical properties and is comprised of at least three distinctly different fibre layers in terms of i) fibre size (i.e. diameter of the fibre), ii) fibre organization or alignment, iii) fibre mechanics in terms of elongation, tensile modulus, and ultimate tensile strength. Specifically, this design is focused on exhibiting decreasing tensile strength, decreasing tensile modulus, with varying fibre organization. As well as exhibiting mechanical properties (compressive and tensile) suitable to provide mechanical integrity of native cartilage, the layered construct of the invention provides a scaffold through which chondrocytes or stem cells can penetrate and remain viable.

<CIT> described a cartilage which is constructed using biodegradable electrospun polymeric scaffolds seeded with chondrocytes or adult mesenchymal stem cells. More particularly engineered cartilage has been prepared where the cartilage has a biodegradable and biocompatible nanofibrous polymer support prepared by electrospinning and a plurality of chondrocytes or mesenchymal stem cells dispersed in the pores of the support. The tissue engineered cartilages of the invention possess compressive strength properties similar to natural cartilage. Methods of preparing engineered tissues, including tissue engineered cartilages, are provided in which an electrospun nanofibrous polymer support is provided, the support is treated with a cell solution and the polymer-cell mixture cultured in a rotating bioreactor to generate the cartilage. The invention provides for the use of the tissue engineered cartilages in the treatment of cartilage degenerative diseases, reconstructive surgery, and cosmetic surgery. <CIT> provides a device for regenerating musculoskeletal tissue having a scaffold comprised of fiber layers adapted to provide mechanical integrity to the scaffold in the form of increased tensile and compressive resistance and one or more other layers adapted to provide mechanical integrity and to provide a suitable biochemical environment.

<CIT> discloses methods of using electrospun fibers to repair and/or regrow hyaline cartilage, e.g. on the articulating surfaces of bones.

The invention relates to a method as disclosed in claim <NUM>. The base of the regeneration construct scaffold is a biodegradable polymer. The biodegradable polymer is blended with natural additives, such as cellulose and hydroxyapatite which increase tissue affinity. The regeneration construct is composed of two layers, where layer I comprises blend of biodegradable polymer and cellulose and is prepared for cartilage tissue formation; and layer II comprises blend of biodegradable polymer, cellulose, and hydroxyapatite and is prepared for subchondral bone tissue formation. The construct has a fibrous morphology that accurately replicates the functions and structure of the natural cartilage. The regeneration construct is composed of polymer fibres providing inner space for deposition of cartilaginous extracellular matrix. The surface of the fibres is chemically modified. A biologically active component, growth factor, incorporated into the construct facilitates the migration, proliferation and differentiation of cells and production of extracellular matrix. After implantation of the regeneration construct, the active components are released over time, resulting in the regeneration of cartilage-like tissue through interactions with host and transplanted stem cells.

A construct for regeneration of articular joint cartilage comprises two-layer scaffold matrix, in which a layer I comprises an ozone-treated scaffold of biodegradable polymers and natural additives; and a layer II comprises a scaffold of biodegradable polymer and natural additives; and the layer I contains embedded biologically active molecules that are slowly and continuously released during operation of the construct.

The layer I and the layer II are made of a biodegradable polymer optionally selected from polycaprolactone, polylactic acid, poly(lactic-co-glycolic acid).

The natural additive of the layer I is cellulose and the natural additive of the layer II is cellulose and hydroxyapatite.

The biologically active molecules encapsulated in the layer I are growth factors which are incorporated to the biodegradable scaffold through functional groups, after the implantation the scaffold starts to degrade, this way releasing the growth factors in a controlled and sustained manner.

The biodegradable polymer is a porous material with a pore size in the range of <NUM> to <NUM>, preferably <NUM> to <NUM>.

The surface of ozone treated regeneration construct layer I scaffold is hydrophilic, with average water contact angle in range from <NUM> to <NUM>°. The regeneration construct layer II scaffold is super hydrophilic, the water contact angle is <NUM>°.

According to the invention, a method of preparation of the regeneration construct is provided, comprising preparation of polymer solutions for layers I and II; preparation of the regeneration construct layer I and II scaffolds by solution cryo-electrospinning; conversion of cellulose acetate to cellulose of the regeneration construct layer I and II scaffolds; surface modification of the regeneration construct layer I scaffold; embedding of biologically active molecules in the regeneration construct layer I scaffold; sterilization of the regeneration construct layer I and II scaffolds; combining of the regeneration construct layer I and II scaffolds in order to form final regeneration construct.

The method of preparation of the regeneration construct additionally comprises drying steps following to (b), (d) and (e) steps.

The biologically active molecules added to the solution of the first layer are growth factors which are bound only on fibers of the regeneration construct layer I scaffold surface. The growth factors are incorporated to the biodegradable scaffold through functional groups. The growth factors stimulate differentiation of stem cells to form chondral or subchondral tissue of layer I and layer II accordingly.

The solvents used for the preparation of polymer blend solutions are polar organic solvents.

The regeneration construct is for use in regenerating cartilage by implantation in a subject in need thereof.

After the implantation of the regeneration cartilage the scaffold starts to degrade, this way releasing the growth factors in a controlled and sustained manner. When released, growth factors stimulate differentiation of stem cells to form chondral or subchondral tissue accordingly. The latter is also stimulated by hydroxyapatite, which is the addition of regeneration construct layer II. After implantation and gradual degradation, regeneration construct withdraws from the defect site, making space for newly formed cartilage and subchondral tissue.

The advantage of the invention in comparison with the already developed methods is a unique sequence of multiple stages of production resulting in the regeneration construct with unique properties.

The articular cartilage regeneration construct is composed of two layers, where a mixture of synthetic and natural polymers (PCL and cellulose) is used to produce the first layer (chondral layer) and the mixture of synthetic and natural polymer (PCL and cellulose) and hydroxyapatite (HAP) as bioactive substance, which is naturally formed in the bone structure is used for the production of the second layer (subchondral layer).

The preparation of the regeneration construct layer I comprises following stages:.

The preparation of the regeneration construct layer II comprises following stages:.

The final regeneration construct is achieved by combining layer I and layer II in order so that layer I is positioned on top of layer II.

Stages of the method of preparation of regeneration construct are schematically presented in <FIG>.

The polymer solutions for regeneration construct layer I scaffold are being prepared by dissolving scaffold polymer (either polycaprolactone (IUPAC: (<NUM>,<NUM>)-Polyoxepan-<NUM>-one, <NPL>) or polylactic acid (IUPAC: poly(<NUM>-hydroxypropanoate),<NPL>), or poly(lactic-co-glycolic acid) (IUPAC: <NUM>-(<NUM>-hydroxyacetyl)oxypropanoic acid, <NPL>) and cellulose acetate (IUPAC name: [(2R,<NUM>,<NUM>,5R,6R)-<NUM>-acetyloxy-<NUM>,<NUM>,<NUM>-trihydroxyoxan-<NUM>-yl]methyl acetate <NPL>) by weight ratio of <NUM>:<NUM>, in solvent mixture of acetone (IUPAC name: propan-<NUM>-one, <NPL>) and N,N-dimethylformamide (IUPAC name N,N-Dimethylmethanamide, <NPL>) by volumetric ratio of <NUM>:<NUM> to obtain polymer solution concentration from <NUM> to <NUM> % (by weight to volume ratio). The preparation of polymers solution is carried out at temperature range from <NUM> to <NUM>, by stirring for <NUM> to <NUM> hours.

The polymer solutions for the regeneration construct layer II scaffold are prepared by dissolving either polycaprolactone or polylactic acid or poly(lactic-co-glycolic acid) and cellulose acetate by weight ratio of <NUM>:<NUM>, in mixture of acetone and N,N-dimethylformamide by volumetric ratio of <NUM>:<NUM>, to obtain polymer solution of concentration from <NUM> to <NUM> % (by weight to volume). The preparation of polymer solution is carried out at temperature from <NUM> to <NUM>, by stirring for <NUM> to <NUM> hours. Hydroxyapatite (IUPAC name Pentacalcium hydroxide triphosphate,<NPL>) is added to the final mixture to obtain concentration from <NUM> to <NUM> % and then continuously stirred at temperature from <NUM> to <NUM>, for <NUM> to <NUM> additional hours to obtain homogeneous solution.

The polymer blend solutions are loaded into a polymer blend solution reservoir. The polymer blend solution is supplied by constant flow rate pump. The polymer solution is supplied continuously into the spinning head at flow rate from <NUM> to <NUM>/h per spinning head for fabrication of regeneration construct layer I scaffold and at flow rate from <NUM> to <NUM>/h per spinning head for preparation of regeneration construct layer II scaffold. Between the spinning head and collector, high voltage is applied to facilitate electrospinning process. During electrospinning process acetone and N, N-dimethylformamide evaporate from polymer fibbers. Grounded rotating drum collector is used for collection of electrospun polymer structure. The temperature of the collector is kept from -<NUM> to -<NUM>. The distance from spinning head and the collector is ranging from <NUM> to <NUM>. High voltage is modulated by a high voltage supply up to <NUM>-<NUM> kV. The temperature of reservoir of polymer blend solution and the supply lines is kept in range from <NUM> to <NUM>. The fabrication environment is maintained within the range of <NUM>-<NUM> for temperature and <NUM>-<NUM> % for relative humidity.

After electrospinning, both regeneration construct layer I scaffolds and regeneration construct layer II scaffolds are placed in a vacuum chamber from <NUM> × <NUM>-<NUM> to <NUM> × <NUM>-<NUM> mPa at the temperature of <NUM> for duration of <NUM> to <NUM>.

The regeneration construct layer I scaffold and regeneration construct layer II scaffold are further immersed in <NUM> to <NUM> NaOH (<NPL>)/water-ethanol (<NPL>) solution at temperature from <NUM> to <NUM>ºC for <NUM>-<NUM> hours. Ethanol concentration is in range from <NUM> to <NUM> vol. After conversion samples are thoroughly rinsed with distilled water until pH value becomes neutral.

The regeneration construct layer I scaffold is immersed in deionized water and treated with ozone, which is bubbled from ozone generator. Ozone generator has an output range from <NUM> to <NUM>/h. The treatment is performed at temperature range from <NUM> to <NUM> for the period <NUM> to <NUM>.

After surface modificationthe regeneration construct layer I scaffold is placed in a vacuum chamber from <NUM> × <NUM>-<NUM> to <NUM> × <NUM>-<NUM> mPa at the temperature of <NUM> for duration of <NUM> to <NUM>.

The growth factors are bound only on fibers of the regeneration construct layer I scaffold surface. The growth factors that could be used are: Transforming growth factor beta-<NUM> (TGFβ-<NUM>), Insulin-like growth factor-<NUM> (IGF-<NUM>), or Bone morphogenetic protein-<NUM> (BMP-<NUM>)). The vial with the growth factor is carefully centrifuged prior to opening and then reconstituted accordingly to the manufacturer's instructions. Direct incorporation is subsequently used to immobilize growth factor per <NUM> to <NUM> diameter of the regeneration construct layer I scaffold.

The regeneration construct layer I scaffold is frozen at temperature range from -<NUM> to - <NUM> for duration of <NUM> to <NUM>, and then freeze dried in vacuum from <NUM> × <NUM>-<NUM> to <NUM> × <NUM>-<NUM> mPa at the temperature of -<NUM> to -<NUM> for duration of <NUM> to <NUM>.

Sterilization of the regeneration construct layer I scaffold and regeneration construct layer II scaffold is initiated with a preconditioning of the samples and is carried out at temperature range from <NUM> to <NUM> for duration of <NUM> to <NUM> hours at relative humidity range from <NUM> to <NUM> %. The sterilization performed by <NUM>% ethylene oxide (IUPC: oxirane) environment at temperature range from <NUM> to <NUM> for the duration of <NUM>-<NUM> minutes.

A method of preparation of the regeneration construct comprises preparation of layer I and layer II and combining layer I and layer II in order so that layer I is positioned on top of layer II.

The regeneration construct scaffold has randomly orientated fibers with interconnected pores. In the regeneration construct layer I scaffold fibers diameter ranges from <NUM> to <NUM>. Intra-fiber porosity is in range from <NUM> to <NUM> % by volume, the average pore diameter is in range from <NUM> to <NUM>. Young's modulus is in range from <NUM> to <NUM> MPa. After the cellulose acetate to cellulose conversion the broad absorption band in the <NUM>-<NUM>-<NUM> range appeared in FTIR spectra due to introduced hydroxyl groups in the polymer chains, confirming cellulose acetate conversion into cellulose. The surface of ozone treated regeneration construct layer I scaffold is hydrophilic, with average water contact angle in range from <NUM> to <NUM>°. The absorption of <NUM> to <NUM> % by mass of Phosphate-Buffered Saline solution is obtained after ozone treatment. The melting point of the regeneration construct layer I scaffold is in range from <NUM> to <NUM>ºC. Growth factor is bound to the scaffold via functional groups. <NUM> to <NUM> ng of growth factor is separately loaded on the regeneration construct layer I scaffold and approximately from <NUM> to <NUM> % of growth factor is released to the culture medium within <NUM> hours after incorporation (<FIG>).

The diameter of the regeneration construct layer II scaffold fibers is <NUM> to <NUM>. Porosity is <NUM> to <NUM> % by volume, with average pore diameter of range from <NUM> to <NUM>. Young's modulus is <NUM> to <NUM> MPa. Hydroxyapatite particles (average diameter range from <NUM> to <NUM>, <NUM>-<NUM>% w/v) are distributed evenly all over the fiber volume. The FTIR absorption band in the regeneration construct layer II at <NUM>-<NUM> corresponded to the POa group in hydroxyapatite, and the band at <NUM>-<NUM> is assigned to the hydroxyl group present in hydroxyapatite. The regeneration construct layer II scaffold is super hydrophilic, the water contact angle is <NUM>°. The regeneration construct layer II scaffold absorbs <NUM> to <NUM> % by mass of Phosphate-Buffered Saline solution. The melting peak of the regeneration construct layer II scaffold is in range from <NUM> to <NUM>ºC.

Claim 1:
A method of preparation of a construct for regeneration of articular joint cartilage, wherein said construct comprises a two-layer scaffold matrix, in which: a layer I comprises a scaffold of biodegradable polymers and natural additives; and a layer II comprises a scaffold of biodegradable polymer and natural additives; wherein the natural additives comprise cellulose, wherein said method comprises:
(a) preparation of polymer solutions for layers I and II;
(b) preparation of the regeneration construct layer I and II scaffolds by solution cryo-electrospinning;
(c) conversion of cellulose acetate to cellulose of the regeneration construct layer I and II scaffolds;
(d) surface modification of the regeneration construct layer I scaffold by treating with ozone;
(e) embedding of biologically active molecules in the regeneration construct layer I scaffold;
(f) sterilization of the regeneration construct layer I and II scaffolds;
(g) combining of the regeneration construct layer I and II scaffolds in order to form final regeneration construct.