RU2524618C1 - Combined bone allograft and method for preparing it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is a combined bone allograft representing a non-demineralised bone block prepared of a donor's spongy bone and containing human type I collagen in the form of a fine-cellular sponge prepared by lyophilisation of the bone block impregnated with a collagen solution, throughout the bone block. What is described is a method for preparing the combined bone allograft providing preparing the non-demineralised bone block of the donor's spongy bone, impregnating the bone block with the human type I collagen solution and conducting lyophilisation under the conditions providing the collagen solution transformation into the fine-cellular sponge throughout the bone block.

EFFECT: biocompatible combined bone allograft possesses the mechanical strength, osteoconductive effect, and stimulates osteogenesis.

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Description

The invention relates to medicine, in particular traumatology-orthopedics, and can be used in the treatment of patients with traumatic bone defects, with non-fused fractures, false joints, fractures with delayed consolidation.

With the development of technological progress, the number of patients who have suffered a high-energy injury is steadily growing. The number of patients with open fractures, multi-fragmented fractures, fractures accompanied by a bone defect is increasing. The imperfection of bone defect repair methods, stimulation of bone regeneration lead to a significant increase in the number of nonunions and false joints. So, from 6 to 51.8% of cases of fractures of the long tubular bones during treatment are complicated by nonunion and development of false joints [O.V. Kudashova, A.N. Turchin, S.A. Tarasov // Application of the biocomposite material “KOLLAPAN” in combination with concentrated platelets for the purpose of early prevention and treatment of nonunion of multi-fragmented fractures of long tubular bones, false joints and osteomyelitis, - Biomaterials, - 2012, - No. 15 p. 3].

One of the treatment options for such patients is the filling of a traumatic bone defect or defect after resection of bone fragments with a non-demineralized spongy bone allograft. Such a graft has mechanical strength, allows you to reposition small fragments on its basis, after which it serves as a support for a sufficiently long period. Non-mineralized bone allograft has a weak osteoinductive effect, the process of bone consolidation is not significantly accelerated. The reason for the lack of an osteostimulating effect is that mesenchymal cells are poorly fixed on the trabecular bone allograft due to its coarse-cellular structure and low biocompatibility. Long biodegradation is due to the large volume of non-demineralized bone tissue. The main advantage of such a transplant is its mechanical strength - the ability to perform a supporting function. However, there is a need to increase the osteoconductive and osteoinductive effect by developing combined bone grafts that simultaneously provide biocompatibility, mechanical strength and accelerate bone regeneration.

A composite (combined) implant (Collap), consisting of synthetic hydroxyapatite, collagen and an antibiotic, which is intended to treat a traumatic bone defect or defect after resection of bone fragments [Pankratov AS, Lekishvili M., can serve as an analogue of the proposed combined bone graft]. V., Kopeckiy I.S. // Bone grafting in dentistry and maxillofacial surgery, - M., - BINOM, - 2011. - p. 128]. The main advantages of the "Valve" is that it has osteoconductive and osteoinductive effects. The components of the “Kollapana” are utilized in the patient’s body, biodegradable, while creating the necessary conditions for enhancing bone tissue repair in the area of the bone defect. There are various forms of release of Kollapan implants: standard granules, gel, plates [A. Snetkov. et al. // Application of collapse implants in pediatric bone pathology, - Biomaterials, - 2012, - №14. p. 4]. The main disadvantage of all forms of release of the "Valve" is the lack of mechanical strength, i.e. it cannot be used as a framework and support for the reposition of small-fragmented fractures.

Prototype. The closest technical solution adopted for the prototype is a non-demineralized spongy bone graft, which is filled with a traumatic bone defect or defect after resection of bone fragments [Bone allografts what they can offer and what they cannot / C. Delloye, O. Comu, V. Druez, O. Barbier // J Bone Joint Surg [Br] 2007; 89-B: 574-9]. The graft has sufficient mechanical strength, i.e. ability to perform a supporting function and osteoconductive effect, i.e. serves as a conductor for the formation of the patient’s own bone.

The main disadvantage of non-demineralized grafts is the lack of an osteostimulating effect, i.e. bone consolidation is not accelerated [Bone allografts what they can offer and what they cannot / C. Delloye, O. Comu, V. Dmez, O. Barbier // J Bone Joint Surg [Br] 2007; 89-B: 574-9]. Biodegradation of such a graft takes a long period of time. Large blocks can be absorbed for more than a year [Tkachenko S.S. Osteosynthesis A guide for doctors. - HER Media. - 2012.272 p.]. The reason for the lack of an osteostimulating effect is that mesenchymal cells are poorly fixed in the graft and cannot differentiate.

In this regard, it is proposed to combine non-demineralized allograft bones with human type 1 collagen, as well as with type 1 collagen in combination with human fibrinolytically active plasma (FAL) or with allo bone crumb.

The objective of the invention is to modify the allogeneic non-demineralized graft in order to increase its biocompatibility to ensure osteoinductive effect.

Achievable technical result is to obtain a biocompatible combined bone graft with mechanical strength, osteoconductive effect and stimulating osteogenesis.

Thus, a combined allogeneic bone graft is proposed, which is a non-demineralized bone block obtained from the spongy bone of a donor and containing human type 1 collagen in the form of a sponge with a fine-mesh structure obtained by freeze-drying a bone block impregnated with a collagen solution throughout the volume of the bone block.

Bone blocks preferably have a size of 3.0 × 3.0 × 1.0 cm or 2.0 × 2.0 × 1.0 cm. The combined bone allograft may further comprise allogeneic bone chips or biologically active substances of fibrinolytically active plasma ( FAP).

A method for producing a combined allogeneic bone graft involves the production of a non-demineralized bone block from the spongy bone of a donor, impregnation of the bone block with a human type 1 collagen solution, followed by lyophilization under conditions ensuring the conversion of the collagen solution into a sponge with a fine mesh structure over the entire volume of the bone block.

Lyophilization can be performed, for example, according to the following program: freezing samples to a temperature of -40 ° C, creating a vacuum, maintaining this temperature for 1 hour, raising the temperature to + 36 ° C and holding the samples for 2 hours at a given temperature.

Bone blocks can be made in the size of 3.0 × 3.0 × 1.0 cm or 2.0 × 2.0 × 1.0 cm.

The bone block can be impregnated with a collagen solution, for example, by drilling through holes with a diameter of 2 mm in different directions of the bone block, followed by immersing it in a 10% human type 1 collagen solution and pumping the collagen solution through these holes with a syringe, without using a needle.

For the manufacture of a graft with a combination of human type 1 collagen and bone powder (bone chips), after impregnation of the bone block with a collagen solution, the block is additionally treated with a suspension of allogeneic bone chips, for example, 10% suspension of bone chips with a size of 75-80 microns, in physiological saline using syringe without the use of a needle, progressive-return movements.

For the manufacture of a graft with a combination of human type 1 collagen and biologically active substances FAP after impregnation of the bone block with a collagen solution, the block is additionally treated with fibrinolytically active plasma, for example, at the rate of 0.5 cm ^{3 of} plasma per 1 cm ^{3 of} bone block.

The following are examples that serve to illustrate some preferred embodiments of the proposed combined bone allograft and method for its preparation.

The method of obtaining is as follows.

A combined bone graft is obtained from a spongy bone harvested from a corpse donor. Bones are procured on the basis of the Federal Law of the Russian Federation dated November 21, 2011 No. 323-ФЗ "On the Basics of Protecting the Health of Citizens in the Russian Federation" after it has been found to be suitable for donation in accordance with applicable laws and regulations in the Russian Federation.

Bone harvesting is carried out in a sterile operating room.

Stage 1. Production of bone graft, quarantine.

The selected bone fragments are immersed in a 20% glycerol solution in a Ringer-Lock solution (1000 ml). After 20 minutes of exposure, the bone fragments are wrapped in a sterile material (sheet, oilcloth) and placed in a low-temperature refrigeration unit at a temperature of -40 ° C until the results of pathological anatomical studies of the donor, the study of the biological safety of donor tissues are obtained.

If the biological hazard of the harvested biomaterial is identified, it is disposed of in accordance with the Sanitary Rules and SanPiN 2.1.7.728-99 "Rules for the collection, storage and disposal of waste from medical facilities" (approved by resolution of the Chief State Sanitary Doctor of the Russian Federation of January 22, 1999 . N 2)

Biomaterial, with confirmed biological safety, is transferred to the next stage of bone graft manufacturing.

Bone fragments are cleaned of tissue and, using an electromechanical saw, they are sawn into bone spongy blocks measuring 3.0 × 3.0 × 1.0 cm and 2.0 × 2.0 × 1.0 cm.

The obtained bone blocks are immersed in a 3% solution of hydrogen peroxide, placed in a vacuum chamber and create a pressure of -2 atm. Next, the bone blocks are washed in 0.9% sodium chloride solution, this process is repeated 3-4 times.

The washed bone blocks are placed in alcohol 96% ethanol for 12 hours for dehydration at room temperature. Next, for degreasing, pour in a mixture of alcohol + ether (in the ratio 1: 1) for 24 hours. This process is repeated one more time. Next, bone blocks are placed in the ether solution for 24 hours. Then they are transferred to a sterile box for drying at a temperature of + 37 + 38 ° C.

Upon completion of this step, fat-free, non-mineralized, sterile bone blocks are prepared, ready for further use.

2 stage. Creating a combined bone allograft.

Bone blocks are impregnated with human type 1 collagen, and to stimulate cell growth by a combination of collagen with fibrinolytically active plasma (FAL) or a combination of collagen with bone crumb size 75-85 microns.

To facilitate the uniform filling of bone blocks with a collagen solution, through holes in diameter of 2 mm are drilled in them in various directions. Then, the bone blocks are immersed in a 10% solution of collagen type 1 of a person and, using a needle, pump collagen through these holes using a syringe.

In order to provide additional growth-promoting effect, the bone blocks impregnated with type 1 collagen are subjected to additional processing.

Bone chips contain BMP protein (Bone Morphogenetic Protein), which stimulates bone regeneration. Bone blocks with collagen are additionally impregnated with a syringe without the use of a needle, with translational-return movements of a 10% solution of bone chips (75-80 microns) in physiological saline.

FAP contains a large number of biologically active substances, including PDGF-BB (platelet platelet growth factor). Bone blocks with collagen are additionally treated with FAP at the rate of 0.5 cm ^{3 of} plasma per 1 cm ^{3 of} graft.

Upon completion of this stage, combined (modified) bone allografts are obtained that are impregnated with human type 1 collagen, human type 1 collagen with FAP or human type 1 collagen with bone crumb.

3 stage. Lyophilization.

To increase the shelf life and simplify the storage conditions for products, combined allogeneic bone grafts (CCAT) are lyophilized according to a special program in automatic freeze drying. The selection of this program was carried out in such a way that collagen turned into a sponge throughout the entire volume of the bone block. The program is a sequential passage through the following steps: freezing samples to a temperature of -40 ° C, creating a vacuum, maintaining this temperature for 1 hour, raising the temperature to + 36 ° C and holding the samples for 2 hours at this temperature.

Lyophilized combined allogeneic bone grafts (CCAT) are prepared, ready for packaging and sterilization.

4th stage. Packaging and sterilization.

Upon completion of the 3 stages, the CCAT are sealed in double plastic polypropylene bags, provided with temporary documentation, which indicate the transplant number, the date of preparation and the shelf life and sent for sterilization with gamma rays. Sterilization is performed at an absorbed dose of 20-25 kGy. At the same time, satellite samples of 0.5 * 0.5 * 0.5 cm in size are sterilized in the KKAT batch for sterility control and bacteriological examination. With a negative result of bacteriological examination, KKAT can be used in clinical practice.

Finished Product Parameters

Receive CCAT dimensions of 3.0 × 3.0 × 1.0 cm or 2.0 × 2.0 × 1.0 cm with collagen type 1 person, with collagen type 1 person and biologically active substances FAL, with collagen type 1 person and bone crumbs.

KKAT with collagen, with collagen and FAL, with collagen and bone chips can withstand a load of 1400-1700 MPa. In this case, the stiffness of native cancellous bone samples was 1700-2000 MPa.

As a result of lyophilization, the collagen solution becomes a sponge with a fine-meshed structure, such a fine-meshed structure creates additional benefits for colonization by cells.

Thus, the graft retains the basic mechanical properties, is able to perform a supporting function, and also have osteoconductive and osteoinductive effects, as illustrated by the following experiment.

Fragments of non-demineralized bone matrices and their modifications were placed in the wells of a 6-well Costar plate. To these fragments, 5 ml of a suspension of human fibroblasts in DMEM medium with 10% Gibco bovine fetal serum was added at a rate of 150 × 103 cells per well. In the control wells with cells was a piece of collagen dressing from type 1 human collagen. Two days later, vitally stained cell cultures were evaluated using a Nicon Eclipse 80i microscope at × 50 and × 200 magnification.

The following samples were examined:

No. 1. Native non-demineralized spongy bone matrix;

No. 2. NDMK cancellous bone matrix with type 1 human collagen;

Number 3. NDMK cancellous bone matrix with type 1 human collagen and fibrinolytically active plasma (FAP);

Number 4. NDMK matrix of cancellous bone with collagen type 1 human with the addition of bone powder.

results

After 2 days of cultivation, human fibroblasts were detected on the surface of samples No. 2, 3 and 4, samples No. 1 did not contain cells. In samples No. 2, the content of attached cells on the bone surface by the end of 2 days was 5-8 thousand / cm ² , in samples No. 3 - 16-18 thousand / cm ² , in samples No. 4 - 5-7 thousand / cm ² , in control wells with collagen - 14-19 thousand / cm ² . At the same time, in all wells, the number of attached cells on the glass (outside the bone) was 18-20 thousand / cm ² .

Conclusion:

1. The studied matrices are biocompatible with human fibroblast culture cells, which is due to the absence of toxic substances in the studied samples.

2. Cell adhesion does not occur on the trabecular bone, due to the absence of specific adhesion ligands in the grafts.

3. In samples No. 2 and 4, cell adhesion on the transplant and their proliferation were observed, which is evidence of the osteoconductive and osteoinductive effect.

4. In the presence of sample No. 3, the proliferative activity of the cell culture was 3 times higher than with samples No. 2 and 4. This demonstrates the pronounced growth-promoting effect of the combined bone allograft with FAP.

Claims (12)

1. A combined allogeneic bone transplant, which is a non-demineralized bone block obtained from the spongy bone of a donor and containing the entire type of human collagen bone block 1 type collagen in the form of a sponge with a fine-mesh structure obtained by freeze-drying a bone block impregnated with a collagen solution. 2. The combined bone allograft according to claim 1, in which the bone block has a size of 3.0 × 3.0 × 1.0 cm 3. The combined bone allograft according to claim 1, in which the bone block has a size of 2.0 × 2.0 × 1.0 cm 4. The combined bone allograft according to any one of claims 1 to 3, additionally containing allogeneic bone chips. 5. The combined bone allograft according to any one of claims 1 to 3, additionally containing biologically active substances of fibrinolytically active plasma (FAP). 6. A method for producing a combined allogeneic bone graft, including the manufacture of a non-demineralized bone block from the spongy bone of a donor, impregnation of the bone block with a human type 1 collagen solution, followed by lyophilization under conditions ensuring the conversion of the collagen solution into a sponge with a fine mesh structure throughout the volume of the bone block. 7. The method according to claim 6, in which bone blocks of size 3.0 × 3.0 × 1.0 cm are made. 8. The method according to claim 6, in which bone blocks of size 2.0 × 2.0 × 1.0 cm are made. 9. The method according to PP.6-8, in which lyophilization is the sequential passage of the following steps: freezing samples to a temperature of -40 ° C, creating a vacuum, maintaining this temperature for 1 hour, raising the temperature to + 36 ° C and holding samples for 2 hours at a given temperature. 10. The method according to claim 6, in which the impregnation of the bone block with a collagen solution is carried out by drilling through holes with a diameter of 2 mm in different directions of the bone block, followed by immersion in a 10% collagen solution of human type 1 and pumping with a syringe, without using a needle, a collagen solution through the indicated holes. 11. The method according to claim 6, in which after impregnation of the bone block with a collagen solution, the block is additionally treated with a 10% suspension of allogeneic bone chips of size 75-80 μm in physiological saline using a syringe without using a needle with translational-return movements. 12. The method according to claim 6, in which after impregnation of the bone block with a collagen solution, additional processing of the block with fibrinolytically active plasma (FAP) is carried out at the rate of 0.5 cm ^{3 of} plasma per 1 cm ^{3 of} bone block.