RU2336841C2 - Method for osteoplasty in experiment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: fraction of fibroblast-like cells is obtained from adipose tissue of subcutaneous layer by way of exsufflation, enzymatic treatment and centrifugation. Then osteal chips is added to the obtained cellular fraction as inductor of osteogenic embryonization. One part of chips is used for four parts of cellular faction. When the mixture is prepared it is injected in the defect area. The method allows quick and qualitative recovery of bony tissue and bearing-mechanical features of the bone due to respective proportion of mixture components as well as immunological safety of the operation due to usage of autologous material.

EFFECT: quick recovery of bony tissue and bearing-mechanical features of bone as well as immunological safety of operation.

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Description

The present invention relates to medicine and can be used in osteology, plastic and reconstructive surgery, dentistry, traumatology and orthopedics.

Osteoplastic surgeries include interventions in which a bone defect is simultaneously replaced by a section of another bone, or conditions are created for the gradual replacement of the defect with newly formed bone tissue. Indications for bone grafting are most often the consequences of bone fractures (false joints, bone defects, traumatic osteomyelitis), congenital and acquired bone deformities, skeletal degenerative lesions, bone defects after radical surgery for tumors and bone tuberculosis. Depending on the biological characteristics, auto-, allo-, xeno- and brephogenic bone grafts are distinguished. According to the type of nutrition, the transplants are blood-supplied and necro-supplied; in connection with the donor bed - free and not free (on the feeding leg).

The disadvantages of transplanting non-blood-supplied autografts are additional trauma to the patient in the donor area, the limited amount of osteoplastic material, its sensitivity to infection, and the relatively slow implantation of the graft into the receiving bed due to the limited participation of its cells in the recovery process. The exceptional technical complexity of the transplantation of blood-supplied autografts limits the application of this method.

In case of plastic surgery on the feeding leg, the bone transplantation area is limited, and the extended access for isolation and transposition of such a flap is associated with additional trauma. Non-free bone grafting according to Ilizarov has a fairly wide range of contraindications and limitations: poor condition of the skin in the area of the defect, osteoporosis and small size of bone fragments, size of the defect more than 8-10 cm, the presence of osteomyelitis, lack of discipline and psychological unpreparedness of the patient for long-term treatment with this method.

Allografts are usually used in the form of bone chips or gravel, provided the blood supply to the receiving bed is good without wound infection. Unlike autoplastic material, the cellular elements of a foreign bone always die during transplantation. Dead bone actually becomes a foreign body, the response to which is determined by differences in the antigenic structure of the proteins that form the transplant substance and the tissue of the receptive bed, as well as the state of the body's immune system (Chaklin V.D., 1971).

The closest analogue is the method for replacing bone defects by autotransplantation of osteogenic bone marrow cells grown outside the body (I. Osepyan et al., 1987). Plastic surgery of a bone defect using this method involves the following steps: 1) obtaining bone marrow trepanate from the ridge of the pelvic bone and transferring it to the laboratory; 2) isolation and reproduction in osteogenic cell cultures for 1-1.5 months; 3) reverse transplantation of cultured cells into the bone defect area of a patient whose bone marrow was used for cultivation.

The disadvantages of the method. Surgical trauma and soreness of the donor zone limits the amount of bone marrow that can be obtained, this stretches the cultivation time to generate a therapeutic cell dose. Traditional cultivation methods limit the clinical value of cells due to their gradual loss of proliferative potential and differentiation ability during ex vivo propagation. There is evidence that, when passivated, transformed cells capable of carcinogenesis in vivo appear.

Objectives of the invention: improving the quality of bone regeneration with the restoration of its supporting mechanical properties, reducing the morbidity and safety of the method, reducing the time of rehabilitation.

The essence of the invention is that adipose tissue obtained from the subcutaneous fat layer by aspiration is subjected to enzymatic treatment with a collagenase solution, centrifuged, bone crumb in a 1: 4 ratio is added to the obtained fraction of fibroblast-like cells as an inducer of osteogenic differentiation, and autologous is introduced immediately after preparation the mixture in the area of the bone defect.

The technical result of the proposed method is the elimination of a bone defect. Moreover, the bone marrow is not the bone marrow, but excess and easily accessible adipose tissue as the source of plastic material - osteogenic cells. It is known that the stromal-vascular fraction of adipose tissue contains true “resting” mesenchymal stem cells that have characteristics similar to those of bone marrow stromal cells: they have high proliferative activity, are capable of self-maintenance and multilinear (including osteogenic) differentiation (Zuk PA et al., 2002; Mizuno H., 2003). In the proposed method, osteogenic transformation (proliferation and differentiation) of the transplanted cell fraction occurs in vivo under the influence of a complex of natural osteoinductors secreted by bone crumbs. When implementing this method, tissue culture is not carried out, which is associated with the risk of infection of the cell population, contamination with foreign material of animal origin from the nutrient medium, unpredictable changes in the properties of cells (Wang Y. et al., 2005). The graft consistency in the form of putty provides the maximum contact area with surrounding tissues, the high penetration rate of the cells of the receptive bed and creates the conditions for the rapid reconstruction of the newly formed bone in accordance with the conditions of mechanical load (there is no resorbable core). Thus, the use of the method allows to reduce the invasiveness of the operation, the risk of complications, the terms of rehabilitation, to improve the patient’s quality of life in a shorter period.

The method is as follows. Under local anesthesia, syringe lipoaspiration is performed. Adipose tissue is intensively washed with sterile saline solution from blood impurities and anesthetic. Enzymatic dissociation of adipose tissue and centrifugation isolate the stromal fraction with a predominance of fibroblast-like cells. Bone chips are obtained by treating the edge of a bone defect. Bone chips are added to the stromal cell fraction in a ratio of 1: 4 and the bone defect is filled with this mixture. The fixation of the "graft" is carried out using the surrounding soft tissues, permeable non-resorbable and resorbable materials.

This method was studied experimentally on a model of a defect of the mandibular bone in 8 guinea pigs of 8-10 months of age with a body weight of 600-800 grams.

Example 1. The type and age of the animal: guinea pig, 8 months. Weight before surgery - 600 g. Description of the operation: under intraperitoneal thiopental anesthesia (40 mg per kg of animal weight) and local anesthesia with 40 ml of 0.25% lidocaine solution with adrenaline (1: 1,000,000), syringe liposuction was performed in the inguinal fat pad in the volume 5 ml The resulting lipoaspirate was washed with saline and exposed to a 0.075% solution of type I collagenase (Collagenase Type I, EUROBIO Biotechnology, France) in sterile phosphate buffer (Dulbecco's phosphate buffered saline without Ca & Mg, 1X solution, sterile solution, EUROBIO Biotechnology, France) in for 30 minutes at 37 ° C. To remove residual enzyme activity, the material was repeatedly washed with phosphate buffer and centrifuged to obtain 0.4 ml of a dense precipitate. Under intraperitoneal thiopental anesthesia and local anesthesia with a 0.25% solution of lidocaine with adrenaline (1: 10000), after removal of the hair in the submandibular region and antiseptic treatment of the surgical field, an incision was made along the lower edge of the angle and body of the lower jaw 2.5 cm long. tissue to the bone and exposed the body and angle of the lower jaw. Muscles were exfoliated on both sides of the bone and a through defect with a diameter of 5 mm was created using an end mill. On the right, the defect was left untouched (control). The cell fraction obtained from lipoaspirate was implanted to the left of the defect, having previously added crumb from 1/4 part of the resected bone to it as an inducer of osteogenic differentiation (experiment). The wound was sutured in layers with catgut 5-0 and polyamide 4-0. After 12 weeks, the animal was withdrawn from the experiment by introducing a lethal dose of thiopental sodium. The musculoskeletal preparation of the lower jaw was fixed in a 10% solution of neutral formalin and a comparative macroscopic, radiological and histological study was performed. Upon examination, the defect in which autologous fibroblast-like cells were implanted was filled with bone-like solid tissue, while the control was filled with fibrous tissue. X-ray on the experiment side, the defect region had almost the same density as the surrounding bone. Microscopy in the tissue of implanted defects identified osteoblasts, osteocytes, bone trabeculae and blood vessels.

Example 2. The type and age of the animal: guinea pig, 10 months. Weight before the operation - 800 g. Description of the operation: under intraperitoneal thiopental anesthesia and local anesthesia of 40 ml with a 0.25% solution of lidocaine with adrenaline (1: 1,000,000), 5 ml syringe lipoaspiration was performed in the inguinal fat pad area. The resulting lipoaspirate was washed with saline and exposed to a 0.075% solution of type I collagenase (Collagenase Type I, EUROBIO Biotechnology, France) in sterile phosphate buffer (Dulbecco's phosphate buffered saline without Ca & Mg, 1X solution, sterile solution, EUROBIO Biotechnology, France) in for 30 minutes at 37 ° C. To remove residual enzyme activity, the material was repeatedly washed with phosphate buffer and centrifuged to obtain 0.6 ml of a dense precipitate. Under intraperitoneal thiopental anesthesia and local anesthesia, an incision was made along the lower edge of the lower jaw 2.5 cm long, the underlying tissues were dissected in layers and an end-to-end defect with a diameter of 5 mm was created in the area of the angle of the lower jaw with an end mill. On the right, the defect was left untouched (control). The cell fraction obtained from lipoaspirate was implanted to the left of the defect, having previously added crumb from 1/4 part of the resected bone to it as an inducer of osteogenic differentiation (experiment). The wound was irrigated with an antibiotic and sutured in layers with catgut 5-0 and polyamide 4-0. After 10 weeks, the animal was withdrawn from the experiment by introducing a lethal dose of thiopental sodium. When examining the extracted mandibular bone on the test side, bone formation was observed in the form of a thin plate completely covering the defect, while on the control side there was a through hole with smooth edges with a diameter of 5 mm filled with a tightened scar muscle. On the radiograph of the symmetrical halves of the lower jaw laid next to each other, a higher density is visible in the area of the implanted defect compared to the control. Microscopy of sections stained with hematoxylin and eosin, according to Van Gieson and Koss, the defect area where the implantation was performed is filled with a mature bone of uneven thickness with a periosteum, bone trabeculae and blood vessels (see drawing, a). On the control side, the bone is presented in the form of two fragments lying on the same axis, between which fibrous tissue is located (see drawing, b).

Thus, the results demonstrate the feasibility of using the proposed method of bone grafting to eliminate bone defects. The method is characterized by low invasiveness, immunological impeccability, oncogenic safety, does not require significant material costs and can be applied in practical medicine.

Literature

1. Osepyan I.A., Chaylakhyan R.K., Garibyan E.S., Ayvazyan V.P. Treatment of non-consolidated fractures, false joints and defects of long bones with transplantation of autologous bone marrow fibroblasts grown in vitro and implanted in the AGM // Orthopedics, traumatology and prosthetics. - 1987 .-- 9. - S. 59-61.

2. Chaklin V.D. Bone grafting. - M .: Medicine, 1971. - 228 p.

3. Mizuno H. Versatility of adipose tissue as a source of stem cells // J Nippon Med Sch. - 2003 - Vol.70 (5) - P.428-431.

4. Wang Y., Huso D.L., Harrington J., Kellner J., Jeong D.K., Turney J., McNiece I.K. Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture // Cytotherapy - 2005 - Vol 7 (6) - P.509-519.

5. Zuk P.A., Zhu M., Ashjan P. et al. Human adipose tissue is a source of multipotent stem cells // Mol. Biol. Cell - 2002 - Vol.13 - P.4279-4295.

Claims (1)

The method of bone grafting in an experiment, including the implantation of fibroblast-like cells, characterized in that the adipose tissue obtained from the subcutaneous fat layer by aspiration is subjected to enzymatic treatment with a collagenase solution, centrifuged, bone crumb is added to the obtained fraction of fibroblast-like cells in the proportion of osteogenic differentiation in the proportion of osteogenic differentiation 1 volume of crumbs per 4 volumes of the cell fraction, and immediately after preparation, an autologous mixture is introduced into the area of the bone defect.

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