LT6309B - Porous three dimensional cellulose based scaffold and method - Google Patents

Abstract

The present invention relates to material engineering and could be applied in odontology or other kind of surgery for bone tissue regeneration in a defect site.The aim of this invention is to prepare a porous three dimensional cellulose-based biocompatible scaffold of good osteoconductive and mechanical properties with the structure like that of a natural bone.To achieve the above object, the present invention provides a composite comprising cellulose and biogenic bone particles at a ratio (w/w) 1:0.12-6.0, wherein the diameter of the particles is 0.01-2000 microns. In a separate case a bone material which comprises inorganic compounds and polysaccharide chitin could be used as a biogenic bone. In a separate case the surface of the scaffold could be coated with collagen, platelet-rich fibrin, various growth factors, therapeutic additives, stem cells. The three dimensional porous scaffold of the natural bone morphology is prepared by inserting biogenic bone particles into a cellulose gel during its formation from cellulose acetate at a ratio 1:0.12-6.0, afterwards loading the gel with an aqueous 10-30 % ethanol solution and freeze-dried or extracted with carbon dioxide at supercritical conditions at 24-70 MPa and 35-80oC temperature. The resulting scaffold could be ground to granules of a desired size and/or the pasta could be prepared.

Description

The invention relates to the field of material engineering and can be used in dentistry or other surgery for bone tissue regeneration at the site of the defect.

Three-dimensional porous cellulose backbone with biocompatibility, good osteoconductive and mechanical properties, obtained by composite of natural polymer - cellulose and biogenic sources of calcium, namely, autogenous (native) bone, allogeneic (other human, other species) bone , xenogeneic (other species of bone) bone particles. The structure of the composite, suitable for bone tissue regeneration, is formed by lyophilization or by the action of a supercritical fluid, preincubated with aqueous alcoholic solutions. May be in the form of blocks or granules, or a paste may be produced.

Bone substitutes are used by dentists to repair bone tissue in the defect area. That might be:

- autogenous substitutes - own bone from another site of the same person. Benefits - Does not trigger the immune system to respond. Disadvantages - extra surgery is needed to remove the bone, often bone is missing and it is difficult to give it the required shape;

- allogeneic bone substitutes - bone of another individual of the same species (i.e. human). Physically suitable but capable of transmitting infectious diseases to another person;

- Xenogeneic bone substitutes are the bone of an animal, usually a young bull, in which the protein and only the mineral remains. Its morphology does not quite match the structure of human bone. It is absorbed too quickly by the body;

- Synthetic bone substitutes. Among them, tricalcium phosphate (TCP) and hydroxyapatite (HA) are the most popular.

Most bone substitutes are in the form of powder or granules (0.5 to 2 mm).

Their main disadvantage is that they often jump into a monolithic, non-vascularized piece. This results in an inadequate structure that crumbles when the metal implant is screwed in,

In order to obtain a porous three-dimensional structure, bone substitute particles of various shapes are mixed and assembled from these by various means of adhesion, compression or sintering. Various bony particles, TCP, HA (Winterbottom

J.M. et al. Implant, a method of making this and a mustache for the treatment of bone defects. Patent No. U.S. Pat. No. 6,478,825 B 1, 02-11-2002) or these minerals together with polymer particles (Giomo T. PLGA / HA hydroxyapatite composite bone grafts and method of making. . Three-dimensional composites are also made by electric spinning (Mei Wei, Fei Peng, Zhi-kang Xu. Electrospun apatite / polymer nano-composite scaffolds. Patent No. US7879093 B2, 2011-02-01) or by rapid prototype production technology (carbon). (Rapid Prototyping) (Sawkins, MJ, et al., 3D Cell and Scaffold Patching Strategies in Tissue Engineering. Recent Patent on Biomedical Engineering, 2013, Vol. 6, No. 1, pp. 3-21; Teoh, SH, et al. . Three-Dimensional Bioresorbable Scaffolds for Tissue Engineering Applications. Patent No. US8071007 B 1, 2011-12-06). Synthetic polymers used: poly (s-caprolactone), poly (L-lactic acid), poly (glycolic acid), poly (D, L-lactic-co-glycolic acid), polyphosphazenes, polypropylene fumarate (Muhammad, IS; Xiaoxue, X) .; Li, L. A Review of Biodegradable Polymeric Materials for Bone Tissue Engineering Applications (Journal of Materials Science, 2009, pp. 5713-5724). Of the synthetic polymers, the most widely used is polyphaprolactone) (Dhandayuthapani, B., et al., Polymeric scaffolds in tissue engineering application: A review. International Journal of Polymer Science, 2011, pp.19-19).

However, synthetic polymers have less biocompatibility than natural polymers, often causing tissue necrosis, and their degradation products, such as glycolic acid and other acidic compounds, can increase local acidity, which can cause tissue damage. In addition, toxic metabolites may also be formed in the body through degradation of polymers. The described methods of preparation are complex and do not provide the required porosity because the particles are adherent or sintered. In addition, an important disadvantage is that calcium sources use synthetic mineral compounds TCP and HA.

The closest prototype is a cellulose-based three-dimensional scaffold whose surface is mineralized in a simulated body fluid (SBF) (Petrauskaite, et al.). Biomimetic mineralization on a macroporous cellulose-based matrix for bone regeneration. BioMed Research International. ISSN 2314-6133. 2013, vol. 2013, pp. 1-9). In this case, the porous backbone is derived from regenerated cellulose gel by lyophilization. A uniformly porous block with a morphology corresponding to the structure of the natural bone is obtained.

One of the main drawbacks of the method is that cellulose is not bioactivated, does not grow with bone tissue, and has poor osteoconductive properties. Although the surface of the skeleton is mineralized in SBF solution to improve these properties, it is coated with synthetic minerals, which always have lower biocompatibility than natural ones. In addition, SBF-e mineralization only covers the surface, and the content of minerals in the composites is up to 12 percent by weight of the cellulose, resulting in a slight improvement in the osteoconductive properties of such a scaffold. In addition, the resulting skeleton exhibits poor mechanical properties (Jungian modulus 4 MPa).

It is an object of the present invention to provide a three-dimensional porous cellulose backbone which is characterized by biocompatibility and good osteoconductive and mechanical properties and structure consistent with natural bone morphology.

This object is achieved by forming a cellulose composite with a particle size of 0.01 to 2000 microns in biogenic bone at a weight ratio of 1: 0.12 to 6.0. In an individual case, the cellulose composite is formed from 0.01 to 2000 micron particles of sepia bone containing mineral compounds and polysaccharide chitin. On a case-by-case basis, the surface of skeletal pores is coated with collagen, platelet-enriched fibrin, various growth factors, therapeutic additives, and stem cells. The three-dimensional porous structure, corresponding to bone morphology, is obtained by forming a cellulose gel with a 1: 0.12-6.0 weight ratio of acetylcellulose solution with biogenic bone particles embedded in it, and then lyophilizing or gel supercritical lyophilization. carbon dioxide at a pressure of 24-70 MPa at 3580 ° C. The resulting block-shaped frame can be ground to a desired size of pellets and / or a paste made from them.

The benefits of the resulting scaffold are the use of natural, non-synthetic components: natural polymer - cellulose and comminuted biogenic bones. Cellulose is non-cytotoxic and has biocompatibility. Its degradation products are non-toxic. Autogenous, allogeneic, or xenogeneic (of any animal) bone can be used as biogenic bone. On a case-by-case basis, shredded sepia bone can be used, which, in addition to mineral compounds, also contains polysaccharide chitin, which has anti-inflammatory, antimicrobial and coagulant properties, which provides additional benefits. Cellulose to crushed bone particle weight ratio in composite: 0.12-6.0.

The results of the invention are illustrated in the drawings. FIG. 1 - 1 is a cross-sectional view of a scaffold fabricated by microcomputer tomography. FIG. 2 is a 3D view of a scaffold fabricated in Example 1 obtained by microcomputer tomography. FIG. Fig. 3 is a cross-sectional view of a skeleton fabricated in Example III obtained by microcomputer tomography. FIG. - A three-dimensional image of the skeleton produced in Example 4L obtained by microcomputer tomography. FIG. 5 - Cell proliferation in scaffolds.

Performance of the way.

The broken bones are injected into a solution of acetylcellulose, which produces a regenerated cellulose gel with embedded bone particles. The particle size of the crushed bone is 0.01-2000 microns, the optimum size is up to 200 microns. The porous structure, corresponding to bone morphology, is formed by lyophilizing a cellulose gel with embedded biogenic bone particles or by exposure to supercritical carbon dioxide at a pressure of 24-70 MPa at 35-80 ° C prior to incubation with 10-30% aqueous alcoholic solutions. A variety of water-soluble alcohols can be used, but ethanol is preferred. The composite has good mechanical properties. The Jungian modulus is at least 8 MPa. The composite morphology (porous structure) is fully consistent with human bone morphology (Table). The pores are suitable in size for vascularization (formation of the vascular network) and cell proliferation. In mice, the composite was found to have a dilated vascular network after 2 weeks. Couples communicate with each other and thus transport nutrients and metabolites.

Table. Structural parameters of manufactured composites and natural bone

Frames obtained according to 1,2,3 examples Structural parameters Skeletal volume share,% Porosity, % Specific surface area, mm-l Moderate thickness of beams, mm Couples diameter, mm No. 1 25th 75 15th 0.20 0.1-1.1 No. 2 27th 73 14th 0.21 0.1-1.1 No. 3 28th 72 19th 0.18 0.2-0.6

Jaw bone (location dependent) 7-49 72-93 9-30 0.12-0.41 0.4-1.7

When investigating the proliferation of human osteoblast lineage cell MG-63, it was found that the produced scaffolds have better osteoconductive properties than the prototype-derived scaffold (Fig. (Fig.5). 5).

Method for evaluation of osteoconductive properties. Human osteoblast line MG-63 cells (ATCCVV CRL_1427 ™) (American Type Collection Culture, USA) were used for the assay. The cultured cells are seeded on samples after having been sterilized by UV irradiation for 24 hours. Samples soaked for 1 hour. in culture medium, place them in 24-well plates (one sample per plate) and place the cell suspension thereon. Frames with seeded cells are stored in a thermostat at 37 ° C and 5% CO ₂ . The effect of the test substance on cell growth is assessed by measuring the amount of DNA at 1, 3 and 7 days. DNA content was determined using the fluorochrome Quant-iTP PicoGreen® (Life Technologies, USA) according to the manufacturer's instructions.

The surface of skeletal pores can be coated with collagen, platelet-enriched fibrin, various growth factors, therapeutic additives, stem cells. The frame is obtained in the form of a block from which implants of the desired shape can be easily cut, granules of the desired size can be milled, and a paste can be formed.

The paste is made by mixing the ground composite with glycerol, polyethylene glycol (molecular weight 400-600) or other hydrogel.

sample g dissolved in 261 ml of acetone-ammonia solution (volume ratio: 1: 0.45), add 15 gallogenic bone beads (granule size not exceeding 200 microns), mix well. The resulting dispersion is blown into vessels of desired volume and shape and retained until a solid gel is formed. The resulting gel is thoroughly washed with distilled water. Subsequently, it is kept in 25% ethanol solution for 24 hours and lyophilized. The Jungian modulus of the composite is 8 MPa. A cross-sectional view of the sample obtained by microcomputer tomography is shown in Figs. 1, and the three-dimensional view of Figs. 2. Cell proliferation scaffold data are presented in Figs. 5.

Sample 1 g of acetylcellulose dissolves in 267 ml of acetone-ammonia solution (volume ratio: 1: 0.5), adds 12 g of crushed (particle size up to 100 microns) sepia bone, mix well. The resulting dispersion is blown into vessels of desired volume and shape and retained until a solid gel is formed. The resulting gel is thoroughly washed with distilled water. Subsequently, it is kept in 20% ethanol solution for 24 hours and lyophilized.

Protein is washed from the crushed bone of cuttlefish before being used. For this purpose, the sepia bone powder is filled with 0.5 M sodium hydroxide solution and stirred for 5 hours. At 80 ° C. It is then thoroughly washed with distilled water, dried and sieved.

The Jungian modulus of the composite is 10 MPa. A cross-sectional view of the sample obtained by microcomputer tomography is shown in Figs. 3, and the three-dimensional view of FIG. 4. Cell proliferation scaffold data are presented in Figs. 5.

Sample g dissolves 10 g of acetylcellulose in 1.5 ml of acetone-ammonia solution (volume ratio: 1: 0.45), adds 12 g of crushed xenogeneic bone (bull) (granules not exceeding 200 microns), mixes well. The resulting dispersion is blown into vessels of desired volume and shape and retained until a solid gel is formed. The resulting gel is thoroughly washed with distilled water and exposed to supercritical carbon dioxide at 80 ° C under a pressure of 30 MPa.

The Jungian modulus of the composite is 20 MPa. Cell proliferation scaffold data are presented in Figs. 5.

example

The surface of the fabricated frame is coated with a layer of collagen. For this, type I collagen of various origins (human, porcine, rat, cow, recombinant) is dissolved in 0.1 M acetic acid according to the procedure provided by Sigma Aldrich, then diluted 10 times with phosphate buffer and titrated with 0.1 M sodium hydroxide to pH 7 Place the frame of the desired shape in a centrifuge tube, fill with collagen solution and centrifuge for 10 min at 4000-6000 rpm. The frame is then lyophilized.

Claims (7)

1. A three-dimensional porous cellulose backbone for bone tissue engineering, characterized by a morphology consistent with natural bone structure, characterized in that it comprises a cellulose composite with biogeneic bone particles of 0.01 to 2000 microns in a weight ratio of 1: 0.12 to 6.0. . 2. The three-dimensional porous cellulose backing according to claim 1, characterized in that it consists of a cellulose composite with particles of cuttlefish bone containing mineral compounds and chitin in the size of 0.01 to 2000 microns. The three-dimensional porous cellulose backing according to claims 1 and 2, characterized in that its surface is coated with collagen, platelet-enriched fibrin, various growth factors, therapeutic additives, stem cells. 4. A method of obtaining a three-dimensional cellular cellulose backbone for tissue engineering from regenerated cellulose gel, characterized in that the gel is formed from a solution of acetylcellulose with biogenic bone particles in a weight ratio of 1: 0.12 to 6.0. 5. A process for the preparation of a three-dimensional cellular cellulose backbone for bone tissue engineering by lyophilizing the regenerated cellulose gel obtained according to claim 4, wherein the cellulose gel is encapsulated with 10-30% aqueous alcoholic solution prior to lyophilization. Process for producing a three-dimensional porous cellulose backbone according to claim 4, characterized in that the cellulose gel with the embedded bone particles is incubated with 10-30% aqueous alcoholic solution and exposed to supercritical carbon dioxide at a pressure of 24-70 MPa at 35-80 ° C. Method for producing a three-dimensional cellular cellulose backing according to claims 4, 5 and 6, characterized in that the block-shaped backing is ground to a desired size and / or a paste is produced therefrom.