RU2416434C1 - Bioengineered structure for bony defect closure and osteogenesis and method for producing said structure - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to medicine, and concerns a bioengineered structure for bony defect closure and osteogenesis which represents a hybrid implant comprising a porous polytetrafluorethylene membrane with a M-Ca-P-C-O-N or M-Ca-C-O-N doped multifunction, biocompatible, non-resorbed coating MBNC, where M is a metal chosen from a line including Ti, Zr, Hf, Nb, Ta, with autogenous or allogenic stromal cells recovered from fatty tissue or bone marrow passed on the surface thereof. ^ EFFECT: invention provides reliable degree of cell population attachment to the surface of the bioengineered structure with high cell culture adhesion and growth. ^ 4 cl, 2 dwg

Description

The invention relates to the field of medicine and medical equipment, in particular to cellular technologies for bioengineering structures for use in maxillofacial, cranial surgery, dentistry, traumatology, and can be used to close bone defects with restoration of bone tissue in them, and also the elimination and treatment of bone defects in various departments of the facial or brain skull or other bones and cartilage or their damaged parts.

The term “bioengineered construct” in medicine has emerged relatively recently and is widely used among practitioners in the field of treating bone defects using cell technology (W. Zhang et al. Reconstructing Mandibular Defects with Autologous Bioengineered Tooth and Bone, based on Science Daily. Apr. 5, 2008, the portal “Eternal Youth” http://www.vechnayamolodost.ru/; U.V. Volpert, O.O.Yanushevich, A.S. Grigoryan, N.N. Malginov, A.I. Volozhin. Healing of bone defects of the lower jaw branch of rabbits under bioengineered structures of titanium and a gold alloy with xenogenic mesench mal stem cells // Dentistry, 2009, №1, p.30).

The authors of the monograph A.S. Grigoryan and A.K. Toporkov, “Problems of the integration of implants in bone tissue” // Technosphere, Moscow, 2007, p. 8, define the term bioengineered constructions as follows: “bioengineered constructions (hybrid implants) are complexes from “non-living” material and “living” matter used to replace lost organ-tissue structures of patients. In this case, the replenishment of the "missing" organ-tissue formation is carried out both due to the specific structure of the "non-living" substrate, and due to the transplanted cellular or tissue component of the bioengineered structure. "

The problems of surgical treatment of bone defects in maxillofacial, cranial surgery, dentistry, especially defects of the flat bones of the skull, including the bones of the cranial vault, are one of the most difficult in their solution and remain relevant at the present stage, therefore, the use of cell technology with the use of a resorbable or non-resorbable base in reconstructive surgery is relevant and necessary.

A device for cranioplasty is known that is used to treat skull defects after trepanation, containing a self-hardening plastic plate with three locking elements made in the form of cotter pins made of porous titanium nickelide, with each cotter pin having a flattened head with spikes and a trapezoidal shaft with a divergent end, cotter pins placed in the body of the plate from the ends, and the rods are oriented in the plane of the plate orthogonally to the line of its edge (SU 1655477 A1, A61B 17/58, 06/15/1991).

A device for endoprosthetics of defects in skull bones is known, consisting of a plate for closing a defect with holes for germination of bone tissue and for fasteners, while the plate is made of oxidized metal with a thickness of 0.15-0.35 mm with the possibility of congruent coating of the defect of the skull bones perforated holes with a diameter of 1.5-2.0 mm with a distance between the centers in the row of 4 mm and between the rows of 3 mm, and the fixing elements made in the form of omega-shaped staples are staggered throughout the plate area from a metal with shape memory, arranged in said holes along the edge of plates (RU 2133113 C1, A61B 17/80, 20.07.1999).

A more advanced device for closing defects of the bones of the cranial vault is proposed in patent RU 2308909 C1, A61F 2/28, 10.27.2007. The device consists of a round or oval plate made of oxidized titanium with perforations 1.5–2.0 mm in diameter staggered across the entire plate area with center spacing in the row of 4 mm and 3 mm between rows for bone growth and for fasteners in the form of omego-shaped metal brackets with shape memory.

However, all of these known devices, laborious in design, do not provide stable osseointegration due to their corrosion and for the same reason they gradually lose the quality of high biocompatibility with bone tissue, which over time leads to rejection of the implant.

A known implant for the surgical treatment of diseases of internal organs, which contains a cell suspension placed in an immuno-isolated receptacle made in the form of a continuous volume of spherical or flattened form of porous titanium nickelide with a transverse pore size of not more than 0.5 microns through which the metabolism of the cell a suspension corresponding to a diseased organ and immune cells do not penetrate, since their sizes exceed the pore sizes (RU 2143867 C1, A61F 2/02, 10.01.2000). The known invention is directed to the treatment of diseases of internal organs and allows to increase the life of the implant due to the high biomechanical compatibility. One of the disadvantages of the implant is the poor surface adhesion of the used cell culture, with its insufficiently stable fixation on the surface of titanium nickelide, which can lead to a gradual reverse diffusion of the culture through the cell channels outside the porous framework. In the known patent there is no information about the use of the claimed design for the restoration of bone defects.

There is a method of immobilizing osteogenic stromal bone marrow stem cells on a porous framework, the essence of which is as follows: the cultivation of osteogenic stromal bone marrow cells is carried out after their isolation with a high degree of purification from related shaped elements from bone marrow aspirate without prior mechanical and enzymatic processing and use microcarriers in a monolayer culture; the surface of the porous skeleton is covered with a double protein hydrophilic osteogenic layer by incubating the preform in a solution of collagen in acetic acid and then in DMEM containing a solution of gelatin and β-glycerophosphate; the porous framework is a biologically inert metal alloy of titanium nickelide with a permeable through porous structure with mesh sizes of 300-400 microns and channels connecting them 25-35 microns in diameter; a suspension of osteogep stromal stem cells purified and grown in monolayer cultures is applied to the porous framework before using ex tempore by diffusion deposition on a hydrophilic permeable surface (RU 2329055 C2, A61K 35/28; C12N 5/08; A61F 2/02, 07/20/2007) . Using the known method, it is possible to create a three-dimensional monoculture of osteogenic stromal bone marrow cells with predetermined parameters of the number and density of shaped elements with prolonged proliferative activity and tissue differentiation to simulate the tissue structure and reparative function of the cancellous bone.

The obtained preform from porous titanium nickelide, impregnated with a cell suspension of osteogenic stromal bone marrow cells, can be classified as an engineering structure with high osteogenic properties. The only drawback of this design is only the limitations of its use in the field of maxillofacial and cranial surgery.

The disadvantage for all metal implants made of titanium, titanium nickelide or alloys: titanium-tantalum-niobium (RU 2302261 C1, A61L 27/04, A61K 6/04, 07/10/2007), titanium-cobalt alloy (RU 2341293 C1, A61L 27 / 04, 27/06, 27/04, A61F 2/28) are their low aesthetic characteristics, since these implants are dark spots under the skin on the head, require rigid mechanical fixation, have a high specific gravity and, due to the high thermal conductivity, lead to hypothermia of the implanted region at subzero temperatures.

The aforementioned patent RU 2329055 C2 is the closest analogue in terms of the “method” object.

The closest analogue to the claimed engineering design in terms of the “substance” object is an implant material (abiological hybrid implant), which includes a base (membrane) made of polytetrafluoroethylene (PTFE) with a porosity of 3.0–40.0% and a surface coating layer with a thickness of at least 50 nm, modified with alloying elements M-Ca-CON, M-Ca-PCON, where M-Ti, Zr, Hf, Nb, Ta (RU 2325191 C1, A61L 27/06; 27/14, 27/56, 05.27.2008 )

This patent describes the characteristics of hybrid PTFE-based implants with a coating and cell culture isolated from skin-muscle fibroblasts of human embryos. In in vitro experiments using luminescent microscopy and scanning electron microscopy (SEM), it was shown that on the surface of a non-resorbable polytetrafluoroethylene coated polymeric material, when these embryonic fibroblasts are cultured on them, these cells adhere and expand.

It also follows from the text of the description of this patent that PTFE implants with a coating have high osseointegration potential, high biocompatibility with living tissues and are non-toxic, which makes it possible to define these coatings as multifunctional, biocompatible, non-resorbable coatings (MBNP).

Based on these data, it can be concluded that the clinical use of porous PTFE with MBNP as an abiological carrier of cell culture in bioengineered structures (hybrid implants) for the surgical treatment of bone defects, in particular facial bones and its arch, is promising.

The most promising ensuring the success of the use of cell technology in the treatment of bone defects are the following principles:

- the use of autogenous stromal (stem) cells (sources - bone marrow, adipose tissue);

- the use of committed stromal (stem) cells;

- the use of autogenous stromal (stem) cells and bone cell precursors as part of bioengineered structures.

The latter area is recognized as the most theoretically sound and promising for the surgical treatment of bone defects.

The objective of the invention is to develop a new type of hybrid implants "bioengineered structures" for closing bone defects with the restoration of bone tissue in them and a method for obtaining the specified design.

The technical result of the invention is to provide a reliable degree of fixation of cultured stromal cells (cell population) on the surface of the bioengineered structure, with a high degree of adhesion and growth of the cell population on a biocompatible carrier.

It should be borne in mind that PTFE itself, being a highly biocompatible non-resorbable plastic material with a high level of integration in the tissue environment, is convenient and lightweight, and besides not subject to corrosion, unlike metal implants, at the same time it cannot provide cell adhesion on its surface, which prevents their cultivation on PTFE implants. This problem is solved by applying the above coating to the surface of PTFE.

The technical result regarding the “substance” object is achieved by the fact that the bioengineered design for closing bone defects with restoration of bone tissue in them is a hybrid implant in the form of a porous polytetrafluoroethylene membrane with a multifunctional, biocompatible, non-resorbable MBNP coating doped with M-Ca-PCON elements or M-Ca-CON, where M is a metal selected from the series including Ti, Zr, Hf, Nb, Ta, on the surface of which autologous or allogeneic stromal cells isolated from adipose tissue are passaged bone marrow.

The base of the implant has a pore size of 200-500 microns. Of metals, the coating mainly contains titanium.

In the part of the “method” object, the technical result is achieved by the fact that the method of obtaining the bioengineered design is that stromal cells are isolated from the adipose tissue of the gastrointestinal tract or the bone marrow of the recipient, including precursors of bone cells, followed by cultivation of the cell population from them, which includes sequentially: washing the tissue in a saline solution with antibiotics, grinding the tissue, incubating in a solution of 0.1% collagenase at 37 ° C and constant stirring for 90 min, inhibition of the excreted enzyme by adding 10% fetal calf serum, separating mature adipocytes by centrifugation at 300 g for 10 min, washing the obtained cell pellet from the enzyme in DMEM (Sigma) containing 10% FCS, filtering the resulting cell suspension through a nylon filter and separating it by centrifugation at 400 g for 30 min at room temperature, triple washing the obtained mononuclear cell fractions as a suspension in DMEM medium, while cell cultures were cultured before first passaging in DMEM medium containing 20% serum tissue, then in DMEM medium containing 10% FCS, after which the SCLC culture is passaged onto the surface of the multifunctional, biocompatible, non-resorbable coating of MBNP hybrid implant, which is a porous polytetrafluoroethylene membrane with pore sizes 200-500 μm, while MBNP is doped with M- Ca-PCON or M-Ca-CON, where M is a metal selected from the series including Ti, Zr, Hf, Nb, Ta, 24 hours after passaging the surface of MBNP, the culture medium is changed to osteogenic and osteogenic stimulation is performed using the following while remaining: DMEM medium 10% FCS, 0.01 M 1,25-dihydroxyvitamin D ₃ (Sigma), 50 pM ascorbate-2-phosphate (Sigma), 10 mM β-glycerophosphate (Sigma), when changing the medium every 3 days , then the cells are cultured in an induction medium for 14 days, after which the resulting bioengineered construct is used as intended.

As a metal, the coating mainly contains titanium.

The following criteria serve as criteria for assessing the adequacy of in vitro bioengineered constructs (hybrid implants) obtained in experiments:

- the growth rate of the culture of cells seeded from the adipose tissue of the recipient (for this invention, the rabbit);

- heterogeneity of cell culture, including bone cell precursors;

- phenotypic characteristics of culture cells (signs of preosteoblastic differentiation).

The development of the method for producing the bioengineered design and its testing were carried out on porous samples (membranes) of a hybrid implant based on porous PTFE with MBNP containing Ti-Ca-CON (PTFE pore size is 200 μm) or Ti-Ca-PCON (PTFE pore size is 500 μm), passaged by the cell population.

In order to determine the efficiency of adhesion and growth of culture cells on the surface of bioengineered structures of both observation groups, the results of an in vitro experiment were evaluated using luminescent microscopy and SEM.

To evaluate osteogenic differentiation, cells on MBNP were planted at a concentration of 2.5 × 10 ⁴ cells / cm ² in 6-well plates and cultured until a confluent layer was reached. Then the culture medium was changed to osteogenic. Cell differentiation was evaluated after 14 days by histological staining for alkaline phosphatase activity (NBT / BCIP Stock solution (Roche Diagnostics), extracellular matrix calcification staining (Alizarin Red S (Sigma)) and immunohistochemical staining for osteogenic protein expression.

To determine the expression of various proteins by cells of the culture, immunohistochemical staining with antibodies was used: antiosteopontin, antiosteocalcin, antiosteonectin (Chemicon).

Cells were washed 3 times with phosphate-buffered saline (PBS), then fixed in a 4% solution of paraformaldehyde (Fluka) for 15 minutes. Then, the cells were washed from the fixative for 50 min in 4 FSB shifts (pH 7.4). Antibodies were diluted with a blocking solution of the following composition: 5% bovine serum albumin (Sigma), 0.1% Trition X-100 (Sigma) in 1M FSB solution at concentrations recommended by the antibody manufacturer. The drugs were incubated with antibodies for 12-16 hours at + 4 ° C. Staining with second antibodies, Alexa-488 or Alexa-546 (Molecular Probe), was performed according to the manufacturer's instructions.

The results of cell cultivation, studied using luminescent microscopy on a bioengineered structure with nanostructured MBNP doped with Ti-Ca-C-O-N or Ti-Ca-P-C-O-N elements 14 days after cultivation in osteogenic medium, showed that the surface of the implants was covered with a biofilm of fibroplast-like cells. In the uncoated sample, there are no cells on the surface of the implant.

Figures 1 and 2 show electron diffraction patterns of SEM bioengineered structures based on porous PTFE with MBNP containing Ti-Ca-CON (pore size of PTFE is 200 μm), figure 1, and with MBNP containing Ti-Ca-PCON (pore size PTFE is 500 μm), FIG. 2, passaged by the cell population.

SEM studies have shown that on the surface of bioengineered structures there are numerous, multi-spliced, expanded fibroblast-like cells endowed with cytological characteristics (Figs. 1 and 2), which show their dense settlement on the surface of the structure. No cells were present on the surface of the control samples without coating.

Control experiments with staining cells for alkaline phosphatase showed that rabbit SLCT after 14 days of cultivation in an osteogenic medium on MBNP bioengineered constructs form multilayered areas where the expression of alkaline phosphatase is enhanced - a positive color for alkaline phosphatase activity.

Immunohistochemical analysis of the expression of specific proteins of osteogenesis revealed that the cells are in the initial stages of differentiation, as the expression of osteopontin and osteonectin was detected. Expression of osteocalcin, a late marker of osteogenic differentiation, was absent.

When using zirconium and other metals indicated in the formula in MBNP, results close to titanium were obtained.

Analysis of the obtained data confirms the technical result of the proposed invention. The set of features characterizing the bioengineered structure determines the positive effect of the cultivation of stromal cells from adipose tissue on the surface of the structure. This conclusion follows from a comparison of the results of cell cultivation (bioengineered design with MBNP doped with Ti-Ca-C-O-N or Ti-Ca-P-C-O-N elements) and the control groups of the experiment: intensive population of the surface of the samples with cells was detected in the main group, it was absent. According to the experimental conditions, the cell culture sown on a bioengineered construct with MBNP was subjected from the second day of cultivation and according to the 14th osteogenetic commit.

To use the in vivo bioengineered construct in animal experiments, the phenotypic characteristics of cell populations on MBNP surface of the construct with bone cell precursors (committed stromal cells) were previously studied.

It is known that bone cell precursors and osteoblasts themselves are endowed with a number of specific phenotypic characteristics (Polezhaev L.V. Replacement of skull defects with regenerating bone // Vopr. Neurokhir. 1982, v.2, 53-56). One of them is the expression of the bone isoenzyme of alkaline phosphatase. In addition, osteopontin, osteonectin, and osteocalcin are detected in progenitor cells of osteogenic differentiation using immunohistochemical methods.

According to the data obtained, signs of expression of both alkaline phosphatase and osteopontin and osteonectin were found in the cells on the surface of the samples, but the reaction to osteocalcin was negative.

Thus, in vitro experiments showed that the bioengineered design of PTFE with MBNP is an adequate basis for the cultivation of stromal cells from adipose tissue, and the osteogenic stimulation method commits these cells to the stage of preosteoblasts, and therefore we can talk about achieving the objective proposed inventions, i.e. A new type of hybrid implant was obtained, named as a bioengineered design that meets the requirements of further biomedical tests in in vivo experiments on a model of experimentally reproduced defects of flat bones, in particular, the cranial vault in animals.

Claims (4)

1. Bioengineering design for closing bone defects with the restoration of bone tissue, which is a hybrid implant in the form of a porous polytetrafluoroethylene membrane with a multifunctional biocompatible non-resorbable coating (MBNP) doped with M-Ca-PCON or M-Ca-CON elements, where M - a metal selected from the series Ti, Zr, Hf, Nb, Ta, on the surface of which autologous or allogeneic stromal cells isolated from adipose tissue or bone marrow are passaged. 2. Bioengineering design according to claim 1, characterized in that the implant has a pore size of 200-500 microns. 3. Bioengineering design according to claim 1, characterized in that the coating as an alloying metal contains mainly titanium. 4. A method of obtaining a bioengineered construct according to claims 1 to 3, which consists in the fact that stromal cells are isolated from the adipose tissue or bone marrow of the recipient, including bone cell precursors, followed by cultivation of the cell population from them, which includes: washing the tissue in saline with antibiotics, tissue grinding, incubation in a solution of 0.1% collagenase at 37 ° C and constant stirring for 90 min, inhibition of the isolated enzyme by adding 10% fetal calf serum, from division of mature adipocytes by centrifugation at 300 g for 10 min, washing the obtained cell pellet from the enzyme in DMEM (Sigma) containing 10% FCS, filtering the resulting cell suspension through a nylon filter and separating it by centrifugation at 400 g for 30 min at room temperature, triple washing the obtained fraction of mononuclear cells in the form of a suspension in DMEM medium, while cell cultures were cultured before first passaging in DMEM medium containing 20% serum, then in DMEM medium containing 10% FCS, after which SKLT pass on the surface of MBNP hybrid implant, which is a porous membrane of polytetrafluoroethylene with pore sizes of 200-500 μm, while MBNP is doped with elements M-Ca-PCON or M-Ca-CON, where M is a metal selected from the series Ti, Zr , Hf, Nb, Ta, 24 hours after passaging the surface of MBNP, the culture medium is changed to osteogenic and osteogenic stimulation is performed using the following composition: DMEM medium, 10% FCS, 0.01 μM 1,25-dihydroxyvitamin D ₃ (Sigma), 50 μM ascorbate-2-phosphate (Sigma), 10 mm β-glycerophosphate (Sigma), when changing the medium every 3 days, then the cells are cultured in an induction medium for 14 days, after which the resulting bioengineered construct is used as intended.

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