Method for faster formation of callus in mammals

FIELD: veterinary science.

SUBSTANCE: multipotent mesenchymal stromal cells (MMSC), previously cultivated under the following gas conditions, are added to the area of fracture: 5% O2, 5% CO2, 90% N2.

EFFECT: invention makes it possible to accelerate process of callus formation.

1 dwg, 1 tbl, 1 ex

 

The invention relates to the field of biotechnology and veterinary medicine, in particular to a method of accelerated formation of callus in mammals.

Currently, the use of cell technologies and tissue engineering to enhance healing and restore the functional activity of the tissue upon injury musculoskeletal increasing interest [1, 4, 8]. Search in this area is mainly in two directions: improvement of media for the delivery of cellular material in the injury area, and a directional change of the properties of the cells introduced into the region of the defect. Change properties of cells can be carried out by introducing additional genetic material [10], and due to the modification of the properties of cells, caused by changing the parameters of their environment [11]. Improving bone regeneration after injection in the region of the defect MSC (multipotent mesenchymal stromal cells) was demonstrated in several studies, the results of which are summarized in reviews[1, 2, 8].

Introduction MSC experimental animals with various injuries, such as acute renal failure [12], bleomycin-induced lung injury [13], cartilage defects [14], bone and cartilage defects [15] and others, leads to better time the ranks of the parameters of tissue regeneration.

It is known that the introduction of bone marrow multipotent mesenchymal stromal cells (MMSC) affects the formation of callus after fracture. Currently, with the rapid development of cellular approaches in regenerative and regenerative medicine, an increasing interest is the possibility of obtaining in a short time sufficiently large number of cellular material due to expansion in vitro of cells obtained from body. Multipotential mesenchymal stromal cells or mesenchymal stromal precursor cells (MSC) are adult stem cells of the organism, which are localized in the bone marrow, adipose tissue, and also in several other tissues and organs. These cells have a high proliferative potential, are self-renewing population of cells, supporting their undifferentiated state, and capable of differentiation into specific types of cells under the action of differencirovannyh incentives. In turn, multipotential MSC determines they perform a unique function of formation, maintenance and repair of tissues, and also lies at the basis of modern technologies of tissue engineering. In this regard, since the opening of these cells Alaprintstyles interest in them is growing steadily, however, an increasing number of works of a hundred is it new questions in relation to the operation of MSC in vivo and in vitro.

Multipotential mesenchymal stromal cells (MMSC), which can be isolated from bone marrow, are currently the most popular for reparation of various tissue defects, due to their high proliferative activity in vitro and the ability to osteogenic, adipogenic, chondrogenic and possibly angiogenic differentiation[1, 2, 3, 4, 5]. The ability MMSC to differentiation in osteogenic direction gave reason to assume that their use in the treatment of fractures and damage large areas of the bones can improve the healing process, which was demonstrated in several studies[6, 7, 8].

Previous authors have shown that the cultivation MSC from the bone marrow of rats in conditions of low oxygen content leads to an increase in their proliferative activity and increased resistance to various damaging factors [5].

The authors have tested cell preparations MMSC, preculturing in terms normoxia (20% O2with 5% CO2, 75% N2) and low oxygen (5% O2with 5% CO290% of N2). We have unexpectedly found that the introduction of MSC cultivated with different content About2has a different influence on the formation of callus in the field of experimental fracture at mlekovita is. Histomorphometrical analysis of callus on the 14th day after surgery showed a significant increase of the thickening factor when using MSK, preculturing under conditions of low oxygen (5% O2with 5% CO290% of N2).

Known way to restore the integrity of the bone when the implant based on the culture of cells containing precursor cells osteogenesis [EN 2240135 C1]. However, this method has several disadvantages. Thus, in accordance with this method requires prior formation of the implant, and the use of inducers of osteogenesis.

The known method of reparative osteogenesis in the introduction of suspension cell xenograft-derived mesenchymal stem cells, including the introduction of mesenchymal stem cells into the area of bone defect that was provided by the acceleration and improvement of the quality of regeneration. On the 90th day of the experiment in all groups in the area of the defect has become a complete bone regeneration and bone was successfully inserted into the bone body [7]. This method is taken as the nearest equivalent. Difference is the use of MSC, preculturing under conditions of low oxygen (5% O2with 5% CO290% of N2), which makes it easier to increase KOs is ing corn.

Examples of specific embodiment of the invention.

In this work, we used the growth medium: α-MEM (ICN, USA) supplemented with 2 mm L-glutamine (Sigma, USA), 1 mm sodium pyruvate, 100 u/ml penicillin and 100 mg/ml streptomycin (Gibco, USA) and 10% fetal bovine serum (FCS) (Hyclone, USA) for cell culture; 20 mM phosphate buffer (FB) (Gibco, USA) for washing cells; 0.02% trypsin solution with 0.05% EDTA (Gibco, USA) to release cells from the substrate.

Isolation and culturing of MSC from the bone marrow of rats was carried out as follows. Cells were isolated from the bone marrow of the femur 1,5 month rats male Wistar rats, as described in [5]. Cultivation of selected MSC conducted in CO2-incubator (20% O2with 5% CO2, 75% N2). After the first passage portion of the cells were placed in CO2incubator (N-MMCK), and the other part - in Multigas incubator, which was supported by the gaseous medium 5% O2with 5% CO2and 90% N2(NUR-MSC).

In experiments to study the regenerative potential of MSC were used cells of the second passage.

For analysis of the growth of cells in culture using the method of videomicroscopy. With the help of microscope Leica DM IL (X10 lens) (Leica, Germany)equipped with a digital camera, image archive and, subsequently, in each vial was analyzed 10 stationary field of view area of 1.0 m is 2every 24 hours, starting from the first day after landing and prior to passage. Cells were counted using image analysis SigmaScan Pro 5.0 Image Analysis Software (SPSS Inc. USA). The number of cell doublings was calculated by the formula:

N=3,32lg(x/x0).

Immunophenotype MMSC on the second passage was characterized using monoclonal antibodies as marker molecules: CD90, CD45, CD54, CD73, CD11b, CD44 (BD Bioscience, USA), and cell viability was assessed by using a set of AnnexinV-FITC Kit (Immunotech, France) at a flow cytometer Epics XL (Beckman Coulter, USA).

Cell preparations.

For preparation of cell preparations MSC were detached from plastic trypsinization, iactiveaware enzyme by adding an environment of growth, after which cells resuspendable in the required amount of culture medium containing no FCS. Transportation and storage of cell preparations was carried out on ice for 2-4 hours at a temperature of +4°C. Before the introduction of cell preparation cells resuspendable and selected the required aliquot.

Experimental model operational fracture of fibula in rats.

In the experiment rats were used male Wistar rats (32 specimens weighing about 300 g). The research program was approved by the Commission SSC RF-IBMP of RAS on biomedical ethics. Anesthesia animals undertake is alas injected into the left gastrocnemius muscle of 0.1% atropine in number, the corresponding weight of the animal (according to manufacturer's instructions). Then in the same muscle was injected "Zoletil-100" in a dose of 8 mg/kg (according to the manufacturer's instructions). After the animal has ceased to respond to irritation, it is recorded, was vestigal coat in the area of the planned surgery, three were treated with 3% iodine and 96% alcohol and were cut in the middle part of the fibula [9].

Experimental animals were divided into 4 groups. Animals of the 1st group conducted an operational fracture (group P) without any subsequent additional impacts. Animals of the 2nd group after the change in the damage zone and adjacent muscle tissue was injected with 0.25 ml of medium growth without FCS (group P+C). Animals of the 3rd and 4th groups directly into the fracture immediately after surgery were administered a suspension of N - or NUR-MSC in a small volume of culture medium. Animals of the 3rd group - 0,25 ml of medium growth without FCS containing 5-105N-MMCK (group P+N-MMCK), rats 4th group - 0,25 ml of medium growth without FCS containing 5-105NUR-MSC (group P+NUR-MSC). After the operation the wound in layers sewn surgical thread. In the pre - and postoperative periods the animals were kept in standard vivarium conditions. In the period of rehabilitation the mobility of the animals was within normal limits.

Histological EN is Liz.

Assessment of bone spur in animals was performed at 14 days after surgery. Rats were decapotable, provided the operated bone and cleansed her from the soft tissues. Separated area of callus and were fixed in 4% paraformaldehyde 0.1 M FB (pH 7.0 to 7.2) with 2.5% sucrose at 4°C. After fixation for 10 days were recalcination in 10% EDTA (pH 7.0 to 7.2). Then the samples were obezvozhivani, poured in histoblast and prepared slices with a thickness of 5 μm. The preparations were stained with hematoxylin-eosin, toluidine blue picrotoxinin. Then the sections were determined amount of bone calluses, cartilage volume and the volume of newly formed bone with the help of the program "MOS-VIDEOLAN", and then calculated the ratio of thickening by the formula: diameter of callus/diameter of Mature bone.

The statistical analysis.

The obtained data were subjected to statistical analysis using T-student test.

Results.

Cultivation under reduced oxygen content has resulted in a twofold increase in proliferative activity MMSC. On the 6th day of cultivation, the number of cell doublings under reduced oxygen content (5%) was was 4.02±0.9, and in normal conditions (20% O2) - 1,9±0,3 that is consistent with previously obtained data [5].

Cultivated N-MMCK and NUR-MSC differed morphologically: crops is x N-MMCK on the first passage was dominated by highly spread cells, and among NUR-MSC was more small elongated cells. (See a drawing. Morphological heterogeneity of MSC in bone marrow of rats in the initial stages of cultivation either in normoxia and hypoxia. Presents two-parameter histograms of the distribution of cell size (y-axis) and granularity (x-axis) for cells 4 of the passage, and micrograph (h) for cells of passage 2 after incubation for 96 hours either in normoxia (a) and hypoxia (b). Immunophenotyping of cell markers that are specific for MMSC: CD90 (and 99.8-100%), CD54 (99,5 reached 98.9%), CD73 (94,70-99,8%), CD44 (96,3-97,0%), showed no difference between N-MMSC and NUR-MSC. These cells were negative for markers CD11b (0,39-1,25%), CD45 (0,35-1,43%). The viability of the N-MMSC and NUR-MSC in the studied cultures also did not differ and was 91.6±2% (4,36% apoptotic cells, 3,98% necrotic cells).

Visual inspection of small tibia bones of experimental animals showed that in rats of all 4 groups at the seat of fracture occurred the formation of spindle-shaped bone spur various sizes, quite firmly fastening the proximal and distal bone fragments.

Histological examination of the field of fracture in animals from the control and experimental groups were allowed to establish that the fusion of bone fragments in all cases was the type of secondary healing. Bone spur was and the endosteal corn, connecting bone fragments and representing fibrous tissue and periosteal corn, including cartilage, beams reticulations bone and fibrous tissue. In all the groups studied in endosteal corn were found in the distal and/or proximal fragments of fibula. The majority of fragments were shifted relative to each other. Beams reticulations bones were placed proximally and distally relative to the cartilage. The surface of trabeculae covered with osteoblasts. Thus, cuts in all the groups studied, it was possible to see lots of fabrics, typical of callus, the ratio of which was different in the two groups.

In group P periosteal callus consisted of an array of cartilage cells and is widely looping reticulations bone. On the border reticulations bone and cartilage of the newly formed bone contained a large number of vessels and osteoclasts. In the center of trabeculae, on the border with cartilage were observed small isogenic groups of cartilage cells. The space between akrotiriani plots bones were filled with fibrous tissue, ingrown side angosta.

In animals of group P+, bone spur in quality did not differ from group P, but quantitative differences were quite significant. The size of callus at the Rys group P+were more compared with group P, mainly due to the increase in volume of the cartilaginous part of the periosteal corn. Vascular network in this group was poorly developed.

In group P+HYP-MMCK dimensions of callus in rats were the largest compared to callus animals of other groups. The increase in the size of callus was due to the growth of cartilage tissue, which is determined by an ingrown side of the inner layers of the periosteum bundles of connective tissue. In addition, connective tissue were found in the thickness of the cartilage in isolated islets. From young bone tissue in the cartilage tissue was infiltrated finger bone growths which was a substitute for inducing cartilage. On the surface of shoots was observed in osteoblasts, and between the processes of the numerous blood vessels. In the young bone trabeculae were often isolated chondrocytes. Endothelina corn, mainly consisted of young cells of bone tissue.

In animals of group P+N-MMCK volume of the cartilage tissue was lower than in rats group N+HYP-MMCK. In General, bone spur was similar to that of the rats in group P+HYP-MMCK and differed only in smaller sizes.

The results of the quantitative sistematicheskogo analysis of bone calluses, which confirmed histological observations presented in the table.

Table
Histomorphometrical analysis of bone calluses in rats with experimental fracture of fibula
Experimental groupThe amount of callus (mm3)The amount of cartilage (mm3)The volume of newly formed bone tissue (mm3)The thickening factor
P10,3±2,31,1±0,75,7±1,83,8±0,7
P+C12,7±3,4*1,9±0,7*5,8±1,63,6±0,6
P+N-MMCK14,6±2,7*1,9±0,8*6,5±2,14,5±0,7*,**
P+HYP-MMCK13,7±2,0*2,4±0,5*,**6,0±1,85,0±1,6*,**
Designation:
* - significant differences p is the group P;
** - significant differences with respect to the group P+S.

As follows from the table, the amount of cartilage in rats in group P+, P+N-MMCK and P+HYP-MMCK compared with the control animals (group P) was significantly increased, which well matches the increase in bone calluses in these same groups of animals. In groups P+N-MMSC and P+HYP-MMCK coefficient thickening exceeded the value thereof as compared with the control group P and group P+C. the coefficient of thickening in rats group N+HYP-MMCK was more than animals P+N-MMCK. As for the volume of newly formed bone tissue, which replaced the cartilage tissue periosteal bone spur, the differences in the two groups was not observed.

Summarizing the data of histological and histomorphometrical studies, we can conclude that in rats with a broken fibula, which was introduced MMSC, size was increased developed a bone spur, and this increase was greater in the group of animals which were injected with MSC grown in a hypoxic environment.

In the result, it was found that MSC, cultured under different gas content, have different effects on the parameters of the healing of bone defects.

Thus, the study demonstrated the effectiveness of call for the possible suspension MMSC, pre-expansion are carried out in vitro under different oxygen content, to enhance the formation of bone and cartilage regenerate at an early stage in the formation of callus in experimental fibula fracture in rats.

Literature

1. Prockop D.J. Further proof of the plasticity of adult stem cells and their role in tissue repair. J. Cell Biol. 2003; 160 [6]: 807-809.

2. Bianco p, Riminucci m, Grothos S., et al. Bone marrow stromal stem cells: nature, biology, and potential application. Stem Cells. 2001; 19: 180-192.

3. Lennon D.P., Edmison J.M., Caplan, A.I. Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. J. Cell Physiol. 2001; 187 [3]: 345-355.

4. Davani S., Marandin A., Mersin N., et al. Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model. Circulation. 2003; 108 [10]: 253-258.

5. Borovkova LB, Anokhin E.B. Influence of hypoxia on stromal precursor cells from the bone marrow of rats in the early stages of cultivation. Bull. the experimental. Biol. and the honey. 2007; 143 (4): 386-389.

6. Kruglyakov PV, Sokolova IM, Zinkova NN. and other Effects of syngeneic mesenchymal stem cells for restoring bone tissue in rats after implantation of demineralized bone matrix. Cytology. 2005; 47 [6]: 466-477.

7. Fatkhutdinov TH, R.A. Goldstein, Poulin A.A., and other features of reparative osteogenesis in the transplantation of mesenchymal stem cells. Bull. the experimental. Biol. and the honey. 2005; 140 [7]: 109-113.

8. De is in the RV, Isaev CENTURIES, Kocsis, A., and other Cellular technologies in traumatology and orthopedics; path development. Cell Transplantology and tissue engineering. 2007; 2 [4]: 18-30.

9. Durnova GI, V. Loginov, Kaplanski A.S. Healing fracture of the fibula in rats with long-term posting. Aviakosm. and ecologist. the honey. 2002; 36 [3]: 52-55.

10. Tsuchida H., Hashimoto J., Crawford, E. et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. Orthop. Res. 2003; 2 [1]: 44-53.

11. Hu X., Yu SP, Fraser J.L., et al. Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J. Thorac. Cardiovasc. Surg. 2008; 135 [4]: 799-808.

12. Morigi I.M, Imberti Century, Zoja C, et al. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. Am. Soc. Nephrol. 2004; 15: 1794-1804.

13. Rojas M., Xu J., Woods Ch. R., et al. Bone Marrow-Derived mesenchymal stem cells in repair of the injured lung. Am. J. Respir. Cell Mol. Biol. 2005; 33: 145-152.

14. Ponticiello M.S., Shingle R., Kadiyala S, et al. Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J. Biomed. Mater. Res. 2000; 52 [2]: 246-55.

15. Im G-L, Kim D-y, Shin J-h, et al. Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow. J. Bone Joint Surg. [Br] 2001; 83-B: 289-94.

The method of formation of callus in a mammal, comprising introducing into the fracture multipotent mesenchymal stromal cells (MMSC), characterized in that MSC precultivation in the following gas conditions: 5% O2with 5% CO290% of N2.



 

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6 dwg, 2 tbl

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

6 cl, 13 dwg, 3 ex

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

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