RU2458709C1 - Bioplastic material - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely combustiology, plastic surgery, cosmetology, and can find application as a bioplastic material for skin defect replacement and regeneration stimulation. In the bioplastic material containing a matrix base made of a native form of hyaluronic acid, a frame of the bioplastic material is prepared by the layered lyophilisation of the matrix material to form a space 0.5 to 10 cm thick with fibres of the native form of hyaluronic acid being directed in the porous order and their cells being enriched with thrombocytic lysate with thrombocyte count 1×10⁹ thromb/ml.

EFFECT: higher effectiveness of deep wound healing and biocompatibility.

1 tbl, 3 dwg

Description

The invention relates to medicine, namely to combustiology, plastic surgery, cosmetology and may find application as a bioplastic material for replacing defects in integumentary tissues and stimulating regeneration.

Recently, based on new data obtained by studying the mechanisms of tissue and organ regeneration in order to restore lost functions, tissue engineering and regenerative medicine are developing intensively. The aim of this direction is the creation of de novo organs and tissues, which is achieved through transplantation of cells on carrier matrices.

A carrier matrix or matrix is a synthetic or biological complex that provides mechanical strength to a structure and its spatial 3-D orientation. The main criteria for a biocompatible matrix to create a tissue engineering construct should be: the absence of cytotoxicity, the maintenance of adhesion, fixation, proliferation and differentiation of cells placed on its surface, the absence of an inflammatory reaction to the material and an immune response, sufficient mechanical strength in accordance with the purpose, bioresorbability by the usual metabolic pathways .

Famous Apligraf (Grafskin) is a plastic biomaterial (Tajima K. Regeneration through nerve allografts in cynomologus monkey (Macaca fascicularis). // J. Bone Joint Surgery. - 1991. - P.172.), Created by Organogenesis (USA), the structure is similar to OrCel - matrix, but in contrast to it additionally contains matrix proteins and cytokines.

However, the above material is inaccessible: it requires special conditions of use and has a high price, even for a European level of solvency.

In addition, the presence of collagen in the material leads to sensitization of immunocompetent cells of the body (the formation of an allergic mood), which reduces its clinical effectiveness. The collagen matrix is not metabolized in the wound (does not dissolve as it heals), which in turn requires dressings. With extensive burns, each dressing is a powerful stress factor that causes pain and requires the use of analgesics.

The prototype of the invention is a bioplastic material (RF patent No. 2367476, published September 20, 2009), which includes a matrix base, moreover, the native form of hyaluronic acid having a fibrous structure in the form of a 0.1% solution prepared at room temperature, from the obtained gel-like mass receive microspheres with a diameter of 100 μm, which polymerize, and form an elastic plate. Bioplastic material contains antimicrobial agents.

According to histological and histochemical studies, a unique fibroarchitectonics of biomaterial is noted, which is as close as possible to the structure of the intercellular substance of native tissues. The presence of a hyaluronic amorphous matrix provides it with high strength and optimal efficiency for the replacement and regeneration of underlying tissues.

However, the above material has clinical limitations for use: the material is ineffective for deep defects in the integumentary tissue when the level of damage is below the basement membrane. This circumstance is due to the maximum thickness of the material (up to 0.5 cm), providing incomplete filling of the wound.

Another disadvantage of bioplastic material is that when studying the biocompatibility of this material in cell culture in vitro, low proliferative (cell multiplication) activity was noted.

The technical result of the proposed bioplastic material is to increase the efficiency of healing of deep wounds and biocompatibility.

The problem is solved in that in a bioplastic material that includes a matrix base, the native form of hyaluronic acid is used as the material, the tissue-engineering structure framework is obtained by layer-by-layer lyophilization of the matrix material to form a volume from 0.5 to 10 cm thick, and the fibers of the native hyaluronic form acids are oriented in a spongy order, the cells of which are enriched in platelet lysate with a platelet count of 1 × 10 ⁹  thrombus / ml.

In Fig.1 shows a cross-section of the bioplastic material in Fig.2 - cell culture of bioplastic material, increased by 50 times, Fig.3 - cell culture of bioplastic material, increased by 100 times.

As a matrix, we used the lyophilized form of hyaluronic acid. The technology of layer-by-layer lyophilization of the hyaluronic acid biopolymer allows the formation of bulk biomaterial of a given thickness in the range from 0.5 to 10 cm. Each layer has a microcellular structure, which, like a “sponge,” connecting to each other, forms the frame structure of many micro labyrinths. Such a bioengineering model creates an effective system of the gradient of osmosis of extracellular fluid, providing an optimal trophic environment for cells. In addition, microcells provide reliable cellular fixation and spatial distribution of cellular elements in the wound tissue defect area, which allows better repair of damaged body tissues. The micrograph of the bioplastic material of the cross section shows the presence of cells in a layered arrangement, which determines its availability for enzymes secreted in the wound process and subsequent reparative histogenesis. This circumstance determines the high biocompatibility of the bioplastic material.

The technology of freeze drying was carried out on classical units for freeze drying of the GZL series (LMPIE). GZL freeze dryers are made in strict accordance with GMP standards. The main components of the installation and all parts in contact with the product (working sublimation chamber, shelves, condenser, shut-off valve) are made of stainless steel (grade AISI 316L), with a surface finish Ra <0.5 μm. Condenser temperature up to 5800 ° С. The temperature range on the shelves is from + 550 ° C to 5420 ° C. The location of the condenser is horizontal or vertical. Range of possible operating temperatures of the condenser: 5800 ° С (during freeze drying), + 1210 ° С (during steam sterilization with the SIP sterilization system). Built-in viewing window with backlight. The curvature of the surface of the shelves is not more than ± 0.1 mm / m. The upper shelf is designed for uniform temperature distribution in the chamber. The distance between the shelves is 100 mm (except for the GZL50.5 model). Water cooling (closed loop). Inert gas bottle capping system. Vacuum pressure in the chamber: without loading: # 1 Pa, working vacuum: 10530 Pa, the vacuum is controlled by the operator. The rate of temperature reduction on the shelf: from 1.5 to 20 ° C, the process is completely controlled by the operator. The accuracy of maintaining the temperature on the shelves: ±, 01 ° С.

An example of obtaining biplastic material.

The initial hyaluronic acid hydrogel (0.5-2% hyaluronic acid solution) is poured into 5 ml in 100 ml vials. Freezing is carried out at -40 ° C for 8 hours. Then vacuum dehydration is carried out at a shelf temperature of -40 ° C. The heating rate at the stage of drying is 4-6 ° C / h. The final temperature of the material is 25 ° C. The total vacuum dehydration time is 15 hours. The resulting substance is placed on a horizontal surface and impregnated with platelet lysate (Platelet Rich Plasma) in a ratio of 5 g of substance - 1 ml (1 × 109 thromb. / Ml). Under sterile conditions, they are kept until a homogeneous bulk material is formed.

Enriched platelet lysate with platelet count (1 × 10) was introduced into the original composition of the developed biplastic material. ⁹  thrombus / ml). This platelet concentration is the maximum possible, since a further increase leads to supersaturation and crystallization with a loss of biological properties. The technology of layer-by-layer lyophilization of hyaluronic acid to obtain bulk biomaterial provides a uniform distribution of the concentration of platelet lysate with the preservation of its biological activity.

Platelet lysate is a mixture of biological mediators (Platelet-Derived Growth Factor, Transforming Growth Factor, Vascular Endothelial Growth Factor, Interlikin -4), which play a leading role in stimulating and regulating wound healing processes in the body and are the first to launch a cascade of cell proliferation. The presence of these substances in the developed biplastic material gives the material the ability to create conditions for the growth and division of cells.

Cultivation was performed for 26 days using standard nutrient media: AMEM (Sigma), 2 mM alanyl-glutamine (Invitrogen) and 10% selected calf serum (Gibco). The nutrient medium changed every 4 days or by a change in pH indicators.

Morphology and morphometry

Video documentation was performed daily in each group and series of experiments. The cell area was estimated using the Axio Observer A1 hardware-software complex. Carl Zeiss, on AxioVision software. Cluster analysis of the culture was carried out using the ImageJ and Image Pro Plus software package; cells were divided into groups based on the pixel density per object.

Cell counting and proliferative activity calculation.

Cell counting was carried out in groups 1, 3 and 4 of all series of experiments, on a Vi-Cell XR hardware complex, Becman Coulter, before sowing and at the end of cultivation. Calculation of proliferative activity was carried out according to the following formulas:

where NH is the number of cells during culture;

NH1 is the number of expanded cells;

Based on the above formula, we obtained data on the number of doublings of the culture during the cultivation period. Using the following formula, we determined the rate of crop doubling (doubling / hour) during the calculated number of doubling.

where Ct is the duration of cultivation, hours .;

Dt is the number of culture doublings.

Immunophenotyping was performed on a FACS Canto flow cytometer, Becton Dickinson, immediately before seeding and after cell removal on day 19 and 26 of cultivation. The following antigens were investigated: CD90,44,45, HLA-ABC, HLA-DR, 73,34, 105,14.

Assessment of migration ability was carried out by video recording of cell growth in the first week of cultivation. Recording was performed for 4 hours in all groups of cells, on the hardware-software complex Axio Observer A1. Carl Zeiss. The selection of moving objects (cells), calculation of trajectories and distances of movement with the construction of graphs were carried out using ImagePro Plus software.

During cultivation, an active increase in the density of colonies is observed due to an increase in adhesive cells of small sizes up to 100% at the periphery. In the immediate vicinity of the material there are small areas of discharge.

The morphological picture is good, the cells are small in size, with clear and even edges, tight intercellular contacts, the cytoplasm is uniform, without inclusions, the nuclei are clear with one or more nucleoli.

The total density of the colonies on day 26 was 100%, with the exception of areas of the location of materials (10-15% plastic). On day 26, cells were removed with trypsin-versene solution, counted and analyzed on a flow cytometer. The number of cells from one culture dish (20.3 cm ² ) amounted to 1,500,000 million cells. The number of doublings is 5.73 doublings of the culture, the doubling rate is 0.01 doubled / hour (0.220 doubled / day).

Microscopy findings

Based on microscopy, there is no evidence of material toxicity. Cells grow well in the presence of material. When co-cultured with lyomatrix, an optimal growth rate is observed, which is obviously due to the presence of platelet lysate. A change in the morphological picture was also revealed - in the first stages of cultivation, the cells had a clearer structure, homogeneous cytoplasm, smooth edges and processes, the cell sizes were slightly smaller than the cells in the control. At the last stages of cultivation, differences in the morphological picture were not visualized.

To assess cell activity, an integral coefficient was introduced (the ratio of cells at the stage of mitosis to the total number of cells). As a result of the study, it was found that growth factors in the structure of the biomaterial significantly increased mitotic activity. The results are shown in the table.

The value of the integral coefficient at time intervals 7 days 15 days 19 days The control 0.15 0.2 0.17 Cytokines 0.3 0.25 0.08

The novelty of the developed bioplastic material is that for the first time a combination of the lyophilized form of hyaluronic acid and biological mediators was used. This combination provides high biocompatibility and reparative potential of the bioplastic material.

It is such a bioengineering organization that creates comfortable trophic conditions for cellular elements migrating to the lesion site. In addition, this bioplastic material must contain unique factors of taxis (migration) and cell growth.

Claims (1)

A bioplastic material including a matrix base, the material of which uses the native form of hyaluronic acid, characterized in that the skeleton of the bioplastic material is obtained by layered lyophilization of the matrix material to form a volume of 0.5 to 10 cm thick, with fibers of the native form of hyaluronic acid oriented in a spongy manner, the cells of which are enriched in platelet lysate with a platelet count of 1 × 10 ⁹ thromb. / ml.

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