RU2375981C1 - Method of treating slowly healing fractures by transplantation of autologic mesenchymal stem cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to traumatology and orthopedics and can be applied for treatment of slowly healing fractures. Tunnelling of bone fragments is performed in such way that all tunnels go through fracture zone. Compressive dressing is applied until bleeding stops and blood clots are formed in tunnels. Into formed in tunnels blood clots injectionally introduced is suspension of population of homogenous by phenotype on 87-99.9% autologic mesenchymal stem cells with cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+, isolated from patient's bone marrow and accumulated in vivo in 10% autologic serum.

EFFECT: method allows to create areas of osteogenesis, ensure possibility to recover adequate bone structure.

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Description

The invention relates to medicine, namely to traumatology and orthopedics, and can be used to treat slowly healing fractures of long tubular bones of various locations.

Slow fusion is determined radiologically, when at the standard term of fusion of a fracture of a certain localization, the formation of all components of bone marrow is not observed, and the fragments at the ends are often porous (transparent), because the process of regional resorption proceeds in them. With conservative treatment and the use of external fixation devices, a distinct pathological mobility at the fracture level is clinically determined.

Of the conservative methods of treating delayed consolidation and pseudoarthrosis, treatment with the introduction of autologous blood of a patient (10-20 ml) is known, whose biological value for regeneration was noted by N.I. Pirogov, in the fracture area, blood injection is repeated 3-4 times in 7-10 days ( VD Chaklin Orthopedics. - M.: MEDGIZ. 1957. - Book I. - C.149). There is also known a method of extracorporeal exposure of a patient to a helium-neon laser and ultraviolet irradiation in the Isold apparatus (Kanykin A.Yu., Anisimov AI, Bely KP, Moskalev VP A method for treating delayed consolidation of tubular fractures bones, patent No. 2169025 from 03.20.1996).

Morphogenetic bone proteins are known to be used to stimulate osteogenesis processes (T.A. Einhorn Clinical Application of Recombinant Human BMPs: Early Experience and Future Development / J. of Bone and Joint Surgery. - 2003, vol. 85-A. - P.82-88).

A known method of treating false joints by injecting a cellular embryonic xenograft into the false joint area while maintaining immobilization with a plaster cast, external fixation apparatus or intramedullary fixation (Belousov V.D., Chobanu A.A., Chobanu F.I. Conservative treatment of long false joints bones (clinical aspects of cell xenoblephoplasty). - Chisinau: “STIINZA”, 1990. - 231 p.). The main disadvantages of the method are possible allergic reactions, xenomaterial rejection, the danger of tumor transformation.

Also in clinical practice, they used to treat the introduction of autologous mesenchymal stem cells isolated from the patient’s bone marrow into the pseudoarthrosis. Bone fusion was achieved (Kolosov N.G., Seledtsov V.I., Golnik V.N., Belogorodtsev S.N., Velichko A.Ya., Shivtsova O.A. Stem cells in traumatology and surgery. // Modern methods treatment of patients with injuries and their complications. / Materials of the All-Russian Scientific and Practical Conference. - Kurgan, 2006. - P.207-209). The disadvantage of this method is that the mesenchymal stem cells for further differentiation into osteoblasts need a surface with highly adhesive properties and an osmotic nutrition medium that is absent in the scar-modified tissues of the false joint, and most of the cells will die or migrate beyond this area.

Compression-distraction osteosynthesis is widely used to stimulate the processes of osteogenesis during fractures, slowing of adhesion and the formation of false joints (Traumatology and Orthopedics. / A guide for doctors. / Edited by Corresponding Member of the Russian Academy of Medical Sciences Shaposhnikova Yu.G. - M., 1997. - T.1. - S.454-481). This method was supplemented by various options for a stimulating effect on osteoreparation, including plasty with demineralized grafts in combination with exposure to an electromagnetic field (Emelyanov V.G., Anisimov A.I., Karptsov V.I., Novoselov K.A. and delayed consolidation of the bones of the lower leg (patent No. 20177461 from 10.25.1990); using transosseous elements and implants with osteoinductive properties (Karlov A.V., Khlusov I.A., Sakurov I.V. Patent No. 2189193 dated March 15, 2001, for the consolidation of non-healing fractures and false joints of tubular bones of the lower extremities) )

A known method of treating delayed consolidation of the tubular bones of the limbs according to the patent of the Russian Federation No. 2189193. The treatment is carried out using an external fixation apparatus after conducting osteoinductive implants proximal and distal to the area of the pathological focus or additionally intramedullary. As osteoinductive implants, calcium phosphate coated implants were used, which allowed increasing the bone mineral density. The goal was to reduce the terms of fusion from 5-6 months to 3.5-2.5 months and increase the mineralization (density) of bone tissue. In the presence of a significant amount of scar tissue in the area of slow fusion or pseudoarthrosis, 1-2 needles made through this zone intramedullary, and especially transosseous elements held outside this zone, do not allow creating conditions for a sufficient number of cellular elements to enter the zone of slow bone formation, and the result of treatment may be negative.

A known method for the treatment of delayed consolidation of fractures of the leg tibia (RF patent No. 20177461), when the limb is fixed with an external fixation device, creates a compression of fragments, a demineralized bone allograft is placed subperiostally, and then a low-intensity electromagnetic field of sound frequency is applied to the non-fusion zone. 5 months after surgery, the formation of corns is completed, and complete consolidation is achieved. In this case, the effect on the zone of slow fusion is very traumatic, in fact, surgical tactics are used that are appropriate for the treatment of the formed pseudoarthrosis. Such an operational trauma during delayed consolidation is somewhat unreasonable, additionally disrupts the blood supply in this area, which leads to fusion in terms typical for the treatment of false joints.

In case of delayed fusion of a femoral fracture, a method for perforating an endostatic callus after drilling a canal from the side of the knee joint, with subsequent intramedullary osteosynthesis (Immamaliev A.S., Balashev B.N., Patent No. 03.208 dated 03.30) is proposed. 92 g.). The method is highly traumatic, in terms of the volume of surgical intervention it approaches operations with a formed false joint. A negative point is arthrotomy of the knee joint, which further contributes to the formation of its contracture.

There is a known method of treating delayed consolidation by tunneling (perforation, drilling fragments), or the Beck method, which consists of introducing a thick needle through 2-3 small skin incisions, with the help of which the ends of the fragments are drilled in different directions through the line of an unbranched fracture, performing from 10 to 40 channels. The smallest fragments of bone tissue and hemorrhage resulting from this, as well as irritation of the plastic properties of bone tissue in some cases contribute to the formation of corns while preserving subsequent limb immobilization (V.D. Chaklin Orthopedics.- M .: MEDGIZ, 1957. - Book I.- C.151; A.V. Kaplan Closed injuries of bones and joints. - M .: Medicine, 1957. - P.68-69).

Closest to the proposed method is the method of creating channels and filling the created channels with bone-plastic material, in particular, a spongy bone graft (Karalin A.N., Ovechkin L.A., Azizova R.Kh. Method for surgical treatment of an overgrown fracture and false joint, - application for invention No. 2003119086 from 06.24.2003). It is difficult to completely fill the perforation holes even with a crushed spongy graft without preliminary processing, and the formation of small cavities is unfavorable for subsequent bone formation. In any case, bone formation will go by replacing the placed transplant with a newly formed bone from the perforation holes. In this case, it is impossible to significantly reduce the consolidation time and to approach the terms of fracture fusion, since the transplant replacement process takes 1.5-2 times longer than the fracture fusion.

The aim of the invention is to increase the effectiveness of the treatment of slow-growing fractures with minimally invasive surgical methods by creating the conditions for organizing additional foci of bone formation in the zone of slow fusion to restore full bone.

This goal is achieved by the fact that autologous mesenchymal stem cells (AMSC) with osteogenic potential are isolated from the bone marrow of the patient; they are increased to the required amount in vitro and introduced into the region of slow fusion after tunneling through the zone of slow fused fracture directly into the performed channels. Before the introduction of the cellular material, a pressure bandage is applied until the bleeding stops and blood clots form in the channels. AMSC is injected after the formation of blood clots, and the introduction is carried out at the exit of the needle from the channel. A population of 87-99.9% phenotype homogeneous AMSC with cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ is injected into blood clots in the form of a suspension in 10% autoserum in an amount of 30-50 million in 3-5 ml of solution. It is this introduction that promotes cell adhesion, their subsequent differentiation and the formation of additional foci of osteogenesis.

Implement the method as follows.

A population of autologous mesenchymal stem cells, 87–99.9% homogeneous in phenotype, is isolated from the patient’s bone marrow: cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+. AMSC are expanded in vitro under laboratory conditions (RF patent No. 2303632). On the day of surgery, a suspension of cells is prepared in a 10% autoserum solution, which is immediately transported to the operating room.

The operation is performed under conduction anesthesia or intravenous anesthesia. Tunneling of bone fragments is performed from 3-6 punctures, and all channels must pass through the zone of slow-growing fracture. For tunneling use a spoke with a diameter of 2 mm and an electric or pneumatic drill. Depending on the shape of the fracture line and its length, from 6 to 12 channels are performed in various directions with the intersection of the fracture zone (Fig. 1). A pressure dressing is applied for 10-15 minutes, waiting for the bleeding to stop and the formation of blood clots in the canal area. After removing the dressing and re-treating the surgical field, a prepared suspension of autologous mesenchymal stem cells in the amount of 30-50 million in 3-5 ml of a 10% autoserum solution is injected into the area of the bone canals. When injected, the needle is punctured by a blood clot formed in the channel and a cell suspension is introduced into the outlet of the needle, thereby avoiding pressure trauma to the cells. The formed blood clot has a loose structure with intersecting fibrin filaments and is a matrix on which cell adhesion can occur with their subsequent differentiation, that is, conditions are created for creating additional foci of bone formation. In addition, the formed blood clot provides the cellular material with sufficient conditions for osmotic nutrition in the first days after administration and grows well in the future by the newly formed vasculature, providing subsequently sufficient blood supply in the consolidation zone. Thus, the introduced cells are involved in the process of osteogenesis, they do not leave, they are not washed out by the blood from the zone of the slowly growing fracture. Their “anchoring” in the place of slow consolidation precisely in the created channels crossing the fracture zone allows overcoming the pathological process of nonunion.

Treatment is carried out with fixation of fragments. In the presence of a stable metal structure, prior to the introduction of AMSC, the existing fixation is retained. In the previous conservative treatment of fracture, the preferred fixation method is extra focal osteosynthesis, which causes minimal additional (operational) trauma and allows for clinical control of consolidation.

The invention is illustrated by the following drawings.

Figure 1 Scheme of the implementation of the tunneling of bone fragments.

Figure 2 Radiographs of the right lower leg of patient D. after stopping distraction (atrophic regenerate).

Figure 3 Radiographs of the right lower leg of patient D. after compression to complete contact of the fragments at the level of osteotomy.

Figure 4 Radiographs of the right lower leg of patient D. 3 months after compression and adaptation of the fragments at the level of osteotomy.

Figure 5 Radiographs of the right lower leg of patient D. 2 weeks after tunneling and the introduction of autologous mesenchymal stem cells.

Fig. 6 Radiographs of the right lower leg of patient D. 1 month after tunneling and the introduction of autologous mesenchymal stem cells.

Fig. 7 Radiographs of the right lower leg of patient D. 6 months after tunneling and administration of autologous mesenchymal stem cells.

Clinical example:

Patient M. Dulkevich, 18 years old, underwent treatment for congenital hypoplasia of the right lower limb with shortening of the right lower leg and right thigh (a total of 7 cm). The limb was lengthened according to Ilizarov at 3 levels (lower third of the thigh, upper and lower third of the tibia). In the process of distraction, the formation of an atrophic regenerate is noted at the level of the lower third of the tibia. The rate of distraction was reduced, then the distraction was stopped (Fig. 2), compression was given until the fragments fully contacted (Fig. 3) in order to achieve fusion of the performed osteotomy. Despite the measures taken after 3 months, an x-ray picture of slow fusion with a tendency to the formation of a false joint was revealed (Fig. 4). After tunneling and the introduction of autologous mesenchymal stem cells according to the presented method, positive dynamics of bone tissue formation was observed already after 2 weeks (Fig. 5), and after a month, fusion was clinically roentgenologically detected (Fig. 6). After a 2-week dynamic test, the external fixation apparatus was dismantled. Subsequently, complete structural adjustment was noted in the region of fusion (Fig. 7).

The method was applied in 6 patients with slowly fused fractures of the tibia and femur. In all cases, fusion was achieved in the period from 1 to 3 months after the manipulation. External fixation devices were dismantled at this time without additional immobilization. Thus, fusion was achieved in terms not only corresponding to the terms of the fusion of fractures of a given localization, but in smaller ones. The reduction of treatment time was due to the formation of additional foci of bone formation at the level of a slowly growing fracture. The difference with other methods is statistically significant.

Claims (4)

1. A method of treating slow-growing fractures, including fixation of bone fragments, their tunneling and transplantation of mesenchymal stem cells, characterized in that after the tunneling of bone fragments, which is carried out in such a way that all channels pass through the fracture zone, a pressure bandage is applied before the introduction of cellular material stopping bleeding and the formation of blood clots in the channels, after which a suspension of a population of phenotype homogeneous is injected into the blood clots formed in the channels in 87-99.9% autologous mesenchymal stem cells with cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ isolated from the patient’s bone marrow and accumulated in vitro in 10% autologous serum. 2. The method according to claim 1, characterized in that the tunneling is carried out by creating 6-12 channels crossing the fracture zone. 3. The method according to claim 1 or 2, characterized in that the number of autologous mesenchymal stem cells is 30-50 million in 3-5 ml of a solution of 10% autoserum. 4. The method according to claim 1 or 3, characterized in that the injection of a suspension of autologous mesenchymal stem cells is carried out by removing the needle from the channel.

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