RU2370227C1 - Method of treating multi-splintered and multiple fractures of long tubular bones - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to traumatology and orthopedics, and is intended for treatment of multi-splintered and multiple fractures of long tubular bones. Reposition of bone fragments and their fixation by means of bone or intramedullary osteosynthesis are carried out. On 10-16 day after operation into fracture zone additionally autologic mesenchymal stem cells are introduced by injection in autologic serum in amount 25-50 mln cells, injection being made at following indices of clinical blood test: leucocytes 4-10*10⁹/l, ESR - 2-20 mm/hour, stab neutrophils - 1-8%. Autologic mesenchymal stem cells are population of live 87-99.9% homogeneous on cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ cells, isolated from patient's bone marrow and accumulated in vitro.

EFFECT: method ensures creation of optimal conditions for reparative osteogenesis, stimulation of reparative osteogenesis processes at early stages of compound fracture treatment and prevention of delayed union and formation of false joints, reduction of traumaticity.

2 dwg, 2 ex, 2 cl, 1 tbl

Description

The invention relates to medicine, namely to traumatology and orthopedics, and can be used to treat multi-fragmented and multiple fractures of long tubular bones of various locations with absolute and relative bone defects.

The problem of optimizing the reparative regeneration of bone after a severe mechanical injury is one of the most urgent problems of traumatology, despite all the achievements of medicine and related sciences, the improvement of osteosynthesis methods and structures for fixing bone fragments. The process of restoring bone integrity after an injury is a complex biological process and depends on many factors that can be divided into general (age, concomitant diseases, endocrine, metabolic rate, nutritional disorders, etc.) and local (fracture nature, quality of reposition and immobilization, the amount of damage to surrounding tissues and a violation of the blood supply to the fragments, etc.). When using traditional methods of treatment for multiple fractures, a slowdown in consolidation or non-healing of fractures with the formation of false joints occurs in 4.8-45% of cases.

A large number (up to 51.6%) of unsatisfactory treatment results are noted. So, according to the Central Medical and Social Expert Commission of Moscow for the period from 2001 to 2005, in the structure of complications leading to disability of individuals with isolated injuries of the lower extremities, violation of the reparative regeneration of bone tissue ranks first (65.4%), and with multiple fractures of the lower leg or femur, primary disability reaches 76.4%.

Thus, one of the main tasks of treating any fracture is to create optimal conditions for reparative osteogenesis. But, even in spite of the ongoing therapeutic measures, especially in patients with multiple and multi-fragmented fractures of long tubular bones, a slower healing of fractures and the formation of false joints are often observed, which leads to impaired limb function and disability. Tubular bones (large bones): tibial, femoral, humeral, radial, ulnar have a certain anatomical structure, a certain blood supply, which causes the regeneration process. Various methods are used to prevent undesirable complications.

Treatment of these fractures can be either conservative or surgical. At different times, various conservative methods were used to stimulate the formation of bone callus (Traumatology and orthopedics. T.1. Edited by Yu.G. Shaposhnikov, Moscow, 1997):

1) Introduction between fragments of 10-20 ml of autologous blood, 5-10 ml of autologous red bone marrow.

2) The use of congestive hyperemia.

3) Physiotherapy, UHF, electrophoresis of calcium salts, anabolic hormones (methandrostenolone, retabolil, etc.).

4) Electrical stimulation with weak currents.

5) Mechanotherapy - Turner's method (striking the fracture area with a wooden hammer).

There are a number of surgical methods aimed at correcting reparative osteogenesis when detecting the presence of signs of slower fusion and in the formation of false joints. These include free bone grafting, microcompression - microdistraction in external fixation devices, the use of blood-supplied transplants (vascularized or revascularized), the use of cell technologies, in particular mesenchymal stem cells (MSCs).

It is known that in experiments on rats with fresh injuries of flat bones (parietal bone), biografts with undifferentiated MSCs were tested, as a result of which the optimal MSC density for treatment was found per 1 cm ^{3 of} graft (Kruglyakov P.V., Sokolova I.B. et al. The effect of syngeneic MSCs on bone restoration during implantation of a demineralized bone matrix. // Cytology. - 2005, T.47 (No. 6). - S.466-477).

Known RF patent No. 2240135, according to which the restoration of bone integrity is carried out using the precursor cells of osteogenesis. But according to a known method of treatment, cells are applied to a three-dimensional allogeneic bone matrix or sponge. Autologous MSCs (AMSCs) were introduced in autologous blood serum, and the cells obtained according to the well-known patent were treated with some osteogenesis inducer (insulin, dexamethasone, glycerophosphate), which is often used in the practice of osteosynthesis. The authors of the known method argue that the introduction of cells into the defect zone without fixation will not allow to create the concentration of cells necessary for bone regeneration, due to their washing out of the defect zone.

Known RF patent No. 230167, which protects a biograft for restoration of cartilage tissue (one of the stages of osteosynthesis). The biograft consists of MSCs and cell cultures, as well as a three-dimensional carrier matrix, i.e. an operation is necessary to form the cavity where the biograft is introduced. The treatment method is traumatic.

Treatment of patients with the injection of AMSK was used in the case of the formation of false joints. / Modern methods of treating patients with injuries and their complications. Conference proceedings, Kurgan, 2006, p.207-209 /. The method indicates that a surface with highly adhesive properties is needed. Since we are already talking about the treatment of the formed false joint, the time after the fracture is a long time and the processes of osteosynthesis take place in different conditions than immediately after the fracture.

The well-known work "The effect of autotransplantation of mesenchymal stem cells on distraction osteogenesis" (Cellular and nanotechnology in biology and medicine. Kurgan, 2007, p.149-150).

It is aimed at optimizing distraction osteogenesis using MSCs. In this work, treatment was carried out with 4-fold injection of MSCs under the control of radiography and computed tomography.

In the described method, the treatment was carried out on already formed bones in order to lengthen them or to replace the formed defects in a “calm” bone. The authors studied the rate of regenerate formation during the monthly introduction of cells.

Widespread in the treatment of multi-fragmented and multiple fractures, which have always been considered complex (regardless of whether they are open or closed), surgical methods, in particular the method of intraosseous fixation. He is referred to for fractures of long tubular bones with significant and difficult to eliminate displacement of fragments (A.V. Kaplan. Technique for the treatment of bone fractures. M., 1948). If with multiple fractures it is possible to compare the fragments in a bloodless way, then they try to carry out the treatment by firm fixation.

In the treatment of multi-fragmented and multiple fractures, various methods of osseous and intramedullary osteosynthesis are used. One of the main principles of surgical (surgical) intervention is low-invasiveness - maximum preservation of the surrounding soft tissues, periosteum, blood vessels and nerves, bone marrow. For this purpose, the tunneling method of setting the plate is widely used without opening the fracture zone and, accordingly, without disturbing the nutrition and innervation of fragments and fragments (Methodical recommendations of the Ministry of Health of the Russian Federation).

After the immobilization of the fragments, the fusion occurs due to the mobilization of reparative factors of the patient's body.

The aim of the invention is to stimulate the processes of reparative osteogenesis in the early stages of the treatment of complex fractures and to prevent delayed fusion and the formation of false joints. Such complications arise when there are factors contributing to the slowdown of consolidation, for example, such as the multiple nature of fractures, multi-fragmented fractures with absolute and relative defects, when a deficiency of stem cells arises in the body as a result of severe trauma (a decrease in their number by 10,000 100,000 times). Stem cells - pluripotent cells are precursors of osteoblasts - cells involved in osteogenesis. Stimulation of osteogenesis is necessary in these difficult cases with a less traumatic method of treatment.

The problem was solved with the help of the addition to the main surgical treatment of introducing into the fracture zone (bone defect) 87-99.9% homogeneous in cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ autologous mesenchymal stem cells (AMSC) in the patient’s autoserum in the amount of 25-50 million depending on the amount of damage and bone defect. Moreover, this introduction is carried out in a disposable, less traumatic injection method. AMSC is not administered immediately after the fracture, but after the completion of the phase of exudative inflammation accompanying the injury. The time for the introduction of AMSC is determined by monitoring the indicators of a clinical blood test. The number of implantable AMSCs is determined by the area of the defect of the cortical layer of the bone by x-ray in 2 perpendicular projections.

The introduction of AMSK to obtain a therapeutic effect must be carried out at a certain stage of bone restoration. The time of administration of AMSK is chosen according to the biochemical parameters of the patient’s blood. The authors found that not only high-quality fusion, but also the acceleration of the fusion process depends on the moment AMSK is introduced into the damage zone. Acceleration of bone marrow formation due to the introduction of AMSC at a certain stage of the consolidation process was discovered by chance, after which the authors began to choose the optimal time when AMSC should be administered after the injury. The introduction of AMSC was carried out without the use of three-dimensional matrices.

In all recent works related to the restoration of bone integrity using MSCs, matrices were used that initiated the differentiation of MSCs into osteoblasts. In the claimed invention, such matrices may be fragments of long tubular bones. And although such fixation of cells in the fracture zone on bone fragments and fragments was not determined, it is precisely with multiple and multi-fragmented fractures of long tubular bones that a positive effect is obtained already from a single injection of cells.

Initially, it was noticed that the introduction of AMSK into the fracture zone after setting the plate for fixing the bone after reposition improves the quality of the bone callus, attention was not paid to the timing of the fusion. Comparing the terms of fusion in patients with a fracture of bones of the 2 lower extremities, we noticed that the terms of fusion depend on the time of administration of AMSK, and it is clearly seen radiographically that in a limb additionally treated with AMSK, restoration of bone integrity occurs in 1.5- 2 times faster than where AMSC was not administered. In the presence of multiple and multi-fragmented fractures of long tubular bones, the biochemical characteristics of the blood are important, according to which the dates for the introduction of AMSC are established. The course of the disease in different patients is different, therefore, the digital characteristics of biochemical blood parameters are very important for deciding at what period of treatment it is necessary to administer AMSC in order to get the best treatment result so that restoration of bone integrity is faster.

The use in the treatment of AMSC in autologous serum leads to both the rapid formation of primary bone marrow and the full restoration of the bone structure with restoration of the bone marrow canal. AMSC was injected in autologous blood serum, and AMSC was obtained by the method according to RF patent No. 2303632, in which the cells are not treated with any osteogenesis inducer (insulin, dexamethasone, β-glycerophosphate), which is often used in the practice of osteosynthesis, for example, known from RF patent No. 2240135.

The population of AMSK obtained by the method according to the patent of the Russian Federation No. 2303632, has an advantage in terms of accumulation of in vitro cell mass for treatment. This is due to the method of isolating inoculum from the patient’s bone marrow and the method of their cultivation, due to which the population of living, 87-99.9% homogeneous phenotype, i.e. by cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ cells (AMSC) are obtained with a high degree of homogeneity.

Before the introduction of AMSC, surgical treatment is performed.

To prevent unwanted complications, the authors propose the following sequence of actions.

1) Closed reposition is performed - comparison of fragments on skeletal traction at the preoperative stage (X-ray control on traction is performed) or manual reposition of fragments during surgery. The main objective of such a reposition is to restore the correct axis of the limb (eliminating the shift in width) and restore the length of the bone without an exact comparison of small fragments. Thus, the fracture zone either does not open, or a small incision of 2.0-3.0 cm is performed to compare fragments, small fragments remain displaced to varying degrees. The periosteum is not separated from the fragments and fragments and, accordingly, their nutrition is not disturbed.

2) At the next stage, through small incisions (3.0 cm) in the proximal or distal fragments under the soft tissues, a simulated metal plate is tunnelled through the bone and screwed to the bone through 0.5-1.0 cm incisions in the projection of the plate openings under X-ray control . Additional fixation of the operated segment with plaster casts is carried out, if necessary, depending on the nature of the fracture and the stability achieved by osteosynthesis.

To obtain AMSC for transplantation in a patient before or during an osteosynthesis operation, bone marrow is harvested by puncture, most often from the ilium. Heparinized punctate in DMEM medium was centrifuged with ficoll to isolate fractions homogeneous in phenotype MSCs, which were then passaged repeatedly in DMEM medium with the addition of 15-22% FBS in vitro until cell mass accumulated in accordance with the area of the injury to be treated.

In the postoperative period, 10-16 days after surgery, after the phase of exudative inflammation subsides, which is determined by the indicators of a clinical blood test (leukocytosis, a shift in the leukocyte formula, ESR) and local status assessment (healing of a postoperative wound, edema, hyperemia, etc.) injected AMSK.

The indicators of the clinical blood test, which determine the time for the injection of AMSK, are characterized by the numbers: leukocytes - 4-10 * 10 ⁹ / l, ESR - 2-20 mm / h, stab neutrophils - 1-8% (see table).

The course of the postoperative period. Indicators Preoperative period Dates after surgery 1-3 days 4-7 days 8-14 days Primary healing Secondary healing Primary healing Secondary healing Primary healing Secondary healing Clinical blood test White blood cells N (4-9 * 10 ⁹ ) 9-13 * 10 ⁹ 9-15 * 10 ⁹ 8-12 * 10 ⁹ 12-20 * 10 ⁹ 5-10 * 10 ⁹ 18-30 * 10 ⁹ Shifting stab neutrophils N (1-6%) 8-12% 10-15% 6-10% 14-20% 4-8% 16-25% ESR N (2-15 mm / h) 20-30 mm / h 25-40 mm / h 15-25 mm / h 30-50 mm / h 10-20 mm / h 40-60 mm / h Body temperature N 36.6 +/- 0.5 ° C 36.6 + 1-1.5 ° C 36.6 + 1-2 ° C 36.6 + 0-0.6 ° C 36.6 + 1.5-2.5 ° C N 36.6 +/- 0.5 ° C 36.6 + 1-2.5 ° C Pain syndrome decreases since injury severe pain severe pain pain reduction preservation or increase of pain pain reduction severe pain Local status, healing of postoperative wounds Edema moderate increased edema increased edema swelling reduction preservation or increase of edema swelling reduction preservation or increase of edema Hyperemia absent moderate moderate hyperemia reduction severe hyperemia absent moderate Hyperthermia absent moderate moderate hyperthermia reduction severe hyperthermia absent moderate Wound discharge - blood or serous discharge blood or serous discharge no discharge, blood or serous discharge purulent discharge absent purulent or serous discharge

AMSK in autologous serum is implanted into the fracture zone by injection under image intensifier tubes. The treatment also takes into account the average periods of bone marrow formation, their changes depending on the influence of the above general and local factors. Fusion of fractures in most victims occurs according to the chondroblastic type, in which 4 stages are distinguished:

1) 1 stage of catabolism (7-10 days).

2) stage 2 of differentiation and proliferation (7-14 days).

3) Stage 3 - the formation of primary bone marrow (2-6 weeks).

4) stage 4 - bone marrow mineralization (2-4 months).

AMSC are implanted into the fracture zone in the 2nd - early 3rd stages of bone callus formation (10-16 days after surgery), when inflammatory processes subside in the fracture zone, enzymatic activity decreases and optimal conditions are created for the proliferation and differentiation of pluripotent stem cells, and the formation of cartilage callus begins acting as a matrix for cells.

After the introduction of AMSK into the fracture site in the implantation zone, the general reactions of the body and local pathological processes in the implantation zone were not observed. In patients with multiple fractures (a fracture of both femurs or tibia), autologous serum AMSK was introduced into the zone of a more severe and unfavorable fracture fusion.

According to the described methodology, 10 patients, 4 patients with multiple fractures and 6 with isolated multi-fragmented fractures of long tubular bones were treated. To assess the results, the X-ray method, computed tomography, and clinical studies were used. In all cases of the use of AMSC, fusion of fractures was achieved without slowing down the fusion and without the formation of false joints. In patients with multiple similar injuries, in the treatment of which the same methods of osteosynthesis were used, AMSC were implanted in one of the damaged segments. It was noted that the introduction of stem cells contributes to advancing bone restoration compared to the other segment - without the use of AMSC - 1.5-2 times, which was expressed in the formation of pronounced bone callus.

It was also noted that 1.5-2 times the average timing of fracture fusion and filling of bone defects was noted when AMSC was introduced into the fracture zone in the treatment of isolated comminuted fractures of the lower leg and hip.

Average terms of fracture fusion:

Localization Merger term Collarbone 4-6 weeks Shoulder blade (body) 6-8 weeks Shoulder (neck) 8-10 weeks Shoulder (diaphysis, neck) 8-10 Shoulder (pineal gland) 10-12 Brush (scaphoid) 8-12 Hand (metacarpals, phalanges) 4-6 Vertebral bodies 4-6 Ribs 4 weeks Thigh (neck) 6-8 months Hip (diaphysis) 5-6 months Thigh (condyles) 3-3.5 months Shin (condyles) 2-3 months Drumstick (diaphysis) 5 months Drumstick (ankle) 2-3 months

Examples of treatment of patients

1) Patient K., 45 years old, was admitted to the clinic on the first day after an injury with a diagnosis of Auto injury. Closed comminuted fracture of both bones in / 3 of the right tibia with displacement. Upon admission to the patient, skeletal traction of the right lower leg was performed. Clinical blood test at admission: hemoglobin - 140 g / l, red blood cells - 3.8 * 10 ¹² / l, white blood cells - 5.3 * 10 ⁹ / l, stab neutrophils - 2%, ESR - 8 mm / h. Considering the preservation of the displacement of fragments on skeletal traction after X-ray control and the multifragmental nature of the fracture, surgery was performed 4 days after admission - blocked intramedullary osteosynthesis of the tibia. During the operation, the fracture zone was not opened. On the 2nd day after admission, the patient was taken 40 ml of bone marrow from the iliac crest for the cultivation of AMSC. The postoperative period was uneventful, postoperative wounds healed by primary intention. Clinical blood test on the 9th day after surgery: hemoglobin - 125 g / l, red blood cells - 3.1 * 10 ¹² / l, white blood cells - 7.4 * 10 ⁹ / l, stab neutrophils - 4%, ESR - 15 mm / h, data indicators indicate the absence of an inflammatory process in the area of a postoperative wound. 10 days after the operation, the patient was injected with 50 million AMSC in the fracture zone (in the zone of the greatest relative bone defect) under X-ray control. After the introduction of AMSC, local and general pathological reactions of the body were not observed.

The treatment data are presented in figure 1. Radiograph No. 1 - using AMSC. A comminuted fracture of both bones in the / 3 tibia with a shift, 8 weeks after surgery (intramedullary metal osteosynthesis of the tibia). The picture shows a pronounced periosteal callus in the implantation zone of AMSC and intermedial callus, fusion of a comminuted fracture in / 3 of the fibula. Radiograph No. 2 - treatment of a similar injury without the use of AMSC. A comminuted fracture of both bones in the / 3 tibia with a shift, 8 weeks after surgery (intramedullary MOS of the tibia with additional fixation of fragments by wire cerclage). The picture shows no clear signs of fusion, the gap between the fragments is not filled with bone tissue, the initial signs of intermedia corns. There are no signs of fusion of m / tibia.

Upon discharge, the patient (treatment with AMSK) was given standard recommendations after such operations. 8 weeks after surgery, X-ray control of the right lower leg was performed. On the x-ray (figure 1, No. 1) there was a pronounced periosteal callus in the implantation zone of AMSC and intermedial callus, fusion of comminuted fracture in / 3 of the fibula.

2) Patient B., 38 years old, was admitted to the clinic on the first day after an injury with a diagnosis of Car injury. Closed comminuted fracture of the bones of both legs in sr / 3 with displacement. Upon admission, the patient was performed skeletal traction of both legs. Clinical blood test at admission: hemoglobin - 116 g / l, red blood cells - 2.8 * 10 ¹² / l, white blood cells - 7.3 * 10 ⁹ / l, stab neutrophils - 3%, ESR - 10 mm / h. Given the multiple and multi-fragmented nature of the fractures, the preservation of the displacement of fragments on skeletal traction after x-ray control, 3 days after admission, surgery was performed - bone osteosynthesis of the tibia of both legs. During the operation, fracture zones were opened due to the impossibility of a closed reposition. On the 2nd day after admission, the patient was sampled 40 ml of bone marrow from the iliac crest for the cultivation of AMSC. The postoperative period was uneventful, postoperative wounds healed by primary intention. Clinical blood test on the 11th day after surgery: hemoglobin - 110 g / l, red blood cells - 2.7 * 10 ¹² / l, white blood cells - 7.6 * 10 ⁹ / l, stab neutrophils - 5%, ESR - 13 mm / h. 12 days after the operation, the patient was injected with 50 million AMSC in the fracture zone of the left tibia (in the zone of the greatest relative bone defect compared to the right limb) under X-ray control. After the introduction of AMSC, local and general pathological reactions of the body were not observed. The treatment results are presented in figure 2.

FIG. 2

- Radiograph No. 3 - treatment using AMSC. Cellular fracture of both bones cf / 3 of the left tibia with displacement. 8 weeks after surgery (bone MOS of the tibia with additional fixation with wire cerclage). The picture shows a pronounced periosteal callus in the implantation zone of AMSC.

- Radiograph No. 4 - treatment without the use of AMSC. Cellular fracture of both bones cf / 3 of the right tibia with displacement. 8 weeks after surgery (bone MOS of the tibia). The picture shows no signs of fusion of the tibia and m / tibia.

When the patient was discharged, standard recommendations were given - bed rest, movement in a wheelchair, exclusion of axial load on both legs. Eight weeks after the operations, X-ray control of both legs was performed, on radiographs a pronounced periosteal callus was noted in the area of AMSK implantation in the left leg. Treatment without the use of AMSC: after 8 weeks there were no signs of fusion of the tibia and tibia of the right tibia - Fig. 2, x-ray. No. 4). Full consolidation of the bones of the left tibia was detected 3 months after the operation, while tibial fusion did not occur in the right tibia, and a false joint was formed there 12 months after the operation.

With a sharp deficit of MSCs, which is fixed during such fractures, it could be assumed that a single injection of cells would not be enough for bone regeneration. However, the structure of the long tubular bones, their structure and, consequently, the structure of their fragments and multiple fragments in the fracture zone create conditions so that the AMSC is not washed out. Bone restoration occurs, and it is due to the AMSK introduced in the autologous serum, which is confirmed by the timing of restoration of the same limbs in the same patient, when one limb was treated with AMSK and the other without. The regenerative capacity of the body is the same, but the conditions for bone consolidation are different.

Claims (2)

1. Surgical method for treating multi-fragmented and multiple fractures of long tubular bones, including reposition of bone fragments and fixation using bone or intramedullary osteosynthesis, characterized in that injection autologous mesenchymal stem cells are additionally introduced into the fracture zone 10-16 days after surgery autologous serum in the amount of 25-50 million cells, and the injection is carried out at the following indicators of a clinical blood test: white blood cells 4-10 · 10 ⁹ / l, ESR 2-20 mm / h, stab neutrophils 1-8%. 2. The treatment method according to claim 1, characterized in that the autologous mesenchymal stem cells are a population of living homogeneous 87-99.9% for cell markers CD 34-; CD 45-; CD 44+; CD 90+; CD 105+; CD 106+ cells that are isolated from the patient’s bone marrow and accumulated in vitro.

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