RU2791230C1 - Method for modeling osteomyelitis of the femoral bone in small laboratory animals - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: it can be used to model femoral osteomyelitis in laboratory animals. To do this, intramedullary culture of Stafilococcus aureus is introduced into the bone. Then the graft is inserted intramedullary, a hook-shaped wire is used as an graft. Further, the bone is stabilized with the help of external fixation by cantilever insertion of a wire and connection of the wires to each other with a self-hardening plastic.

EFFECT: invention provides modelling of osteomyelitis of the femur in small laboratory animals under conditions of external hardware fixation of the graft with obtaining the same type of clinical, radiological, histomorphological and laboratory signs.

1 cl, 4 dwg, 1 tbl

Description

The field of technology.

The invention relates to the field of experimental medicine, namely, to methods for obtaining disease models and can be used to model osteomyelitis in laboratory animals.

There are various methods for modeling osteomyelitis in experimental animals based on bacterial inoculation and additional manipulations with tissues. To induce infection, sclerosing agents, fibrin glue, sealants, and the introduction of foreign bodies (lavsan, quartz sand, metal implants) are additionally used. Models are also known using pre-colonized implants in the absence of a bacterial suspension at the surgical site.

Known methods for modeling osteomyelitis, consisting in the introduction of a bacterial suspension into the medullary canal with a preliminary or subsequent installation of an intramedullary metal implant (RU 2 233 486 C1, EN 2 129 735 C1, Harrasser, N. et al. (2016), Lucke M. et al. (2003), Jensen L. K. et al (2017), Hill, P. F. et al (2002), Stewart S. et al (2012).

There are known methods for modeling osteomyelitis using an implant material previously contaminated with a strain of a pathogenic microorganism (Hamel A. et al. (2008), Lei M. et al. (2017), Zhang X. et al. (2017).

Known two-stage methods of modeling osteomyelitis, which consists in the introduction of infectious material into the area of pre-formed ischemic necrosis (RU 2015 153 229 A, EN 2 622 3696 C1).

Known methods do not provide minimal tissue trauma and a stable position of the implant in the intraosseous canal during the development of the infectious process. Simulation of osteomyelitis of the femur in small laboratory animals under conditions of external hardware fixation of the implant with obtaining the same type of clinical, radiological, histomorphological and laboratory signs is not provided.

A known method for modeling acute intramedullary infection of the tibia in rabbits Odekerken J. C. E. et al. (2013), which consists in cotaminating the bone marrow canal with a bacterial strain, inserting a 20 mm titanium rod into it through a transpatellar approach, and closing the defect with bone wax.

The disadvantage of this method is the additional trauma to the patient's own patellar ligament.

Known model Cobb L. H. et al. (2019), which consists in creating a bicortical defect with a diameter of 1.2 mm in rats in the middle third of the diaphysis and implanting an orthopedic screw contaminated with a bacterial strain into it.

The disadvantage of this method is the risk of instability of the orthopedic screw in the progression of the osteomyelitic process.

The essence of the invention.

The task is to create a method for modeling osteomyelitis in small laboratory animals with a stably fixed implant in a minimally invasive surgical intervention.

The method expands the arsenal of known methods for modeling femoral osteomyelitis in small laboratory animals, while minimizing tissue trauma and ensuring a stable position of the implant in the intraosseous canal during the development of an infectious process, which improves the quality of the model. Simulation of osteomyelitis of the femur in small laboratory animals under conditions of external hardware fixation of the implant is provided with obtaining the same type of clinical, radiological, histomorphological and laboratory signs.

The technical result is achieved by the fact that in the method of modeling osteomyelitis of the femur in small laboratory animals, a culture of Staphylococcus aureus is intramedullarily injected through a burr hole into the bone, then an implant is introduced intramedullary into the bone, a hook-shaped curved wire is used as an implant, the bone is stabilized by external fixation, by console introduction of the spokes and connecting the spokes to each other with self-hardening plastic.

The invention is illustrated by images:

fig. 1 - shows the stages of surgical intervention, where the photo shows: a - a burr hole is made in the femoral diaphysis, b - the S.aureus culture is introduced into the bone marrow canal, c - the implant is inserted (the needle is inserted into the bone marrow canal), d - the external type of implant (intramedullary wire), e - shows the sutured surgical wound, the cantilever wire; e - shows the connection of the spokes with self-hardening plastic to provide external fixation.

fig. 2 - external view of the animal at the stage of the experiment. Separation of purulent exudate from the spoke channels.

fig. 3 - paraffin histotopographic sections are shown, which show a fibrous-abscessed form of osteomyelitis in the metadiaphyseal part of the femur of rats:

in photo a - paraffin histotopographic sections. Trichrome stain according to Masson. SW. ×10;

photo b - fibrinoid necrosis of the central part of the osteomyelitic cavity. Paraffin sections. Gram stain. SW. ×400

fig. 4 shows paraffin sections. Connective tissue sheath cavity:

a) microabscesses in the area of modeling osteomyelitic lesions of the femur of rats. paraffin sections. Staining with hematoxylin and eosin. SW. ×200;

b - inflammatory cell (mainly neutrophilic) infiltration of the area of microabscess formation. paraffin sections. Staining with azure-eosin according to Maksimov. SW. ×1000;

c) Staphylococcus aureus bacterial colonies on the surface of bone sequesters. paraffin sections. Gram stain. SW. ×1000

Implementation of the invention.

A method for modeling femoral osteomyelitis in small laboratory animals consists in intramedullary introduction of a culture of Stafilococcus aureus into the bone, then the implant is inserted intramedullarily and the implant is stabilized using an external fixation device.

Under operating conditions, under general anesthesia, access to the anterior surface of the femur is performed through a vertical incision of the skin and subcutaneous tissue. Then, a trepanation of the diaphysis of the bone is performed at the border of the upper and middle thirds (Fig. 1, a). Next, a culture of Stafilococcus aureus (museum strain ATCC 29213) is injected intramedullary in a volume of 50 μl, the concentration of microorganisms was ¹⁰⁸ CFU/ml. The culture is injected with an insulin syringe, completely deepening the needle into the medullary canal (Fig. 1, b).

At the next stage, an implant is introduced into the medullary canal - a hook-shaped wire with a diameter of 1.0 mm, to a depth of 7 mm and tightly, the surgical wound was sutured in layers with 4/0 vicryl (Fig. 1, c, d, e). The final stage at the level of the lower third of the femur is the cantilever insertion of a spoke with a diameter of 0.6 mm and the connection of the spokes to each other by the method of reinforcement and pouring with self-hardening plastic (Fig. 1, e, f). In the postoperative period, antimicrobial agents are not used in animals, the treatment of knitting needles and the surgical wound is not carried out.

In the course of observations, on average for 9-10 days, all animals noted the release of purulent exudate from the spoke channels. Bacteriological examination of exudate from the intermedullary canal showed the presence of S. aureus in 100% of cases (Fig. 2). X-ray studies showed that by the 15th day after the operation, the animals developed signs of inflammation, limited to the intermedullary space and manifested by the presence of faintly noticeable chaotic rounded areas of enlightenment. By the 21st day of the experiment, the inflammatory process spread to all structural elements of the bone, which was characterized by the presence of small focal cavities in the medullary canal, as well as signs of destruction of the cortical plate and resorption of the cancellous bone of the distal metaphysis. Histological examination of the implantation area in animals in the middle part of the femoral shaft on the 21st day after the operation revealed an extensive osteomyelitic cavity (Fig. 3, a) with purulent-hemorrhagic contents and fibrinoid detritus, including microcolonies of S. aureus (Fig. 3, b).

The osteomyelitic cavity was limited by a fibrous membrane, the inner layer of which was reactively altered loose connective and granulation tissues infiltrated with inflammatory cellular elements with a predominance of neutrophilic granulocytes. The extracellular matrix of the outer layers of the connective tissue membrane included large bundles of concentrically oriented collagen fibers. In the composition of the outer part of the fibrous membrane, microabscesses were found, which are rounded inclusions of reactively altered loose connective tissue (Fig. 4a). They were distinguished by a high concentration of polymorphonuclear leukocytes (Fig. 4, b) and centrally located microcolonies of staphylococci or bone sequesters infected by them (Fig. 4, c).

The bone membrane of the osteomyelitic cavity, or involucrum, was formed by a finely looped network of endostally formed coarse fibrous trabeculae. The intertrabecular spaces were filled with reactively altered loose connective tissue. In the areas of the cortical plate adjacent to the osteomyelitic cavity, there were acellular zones actively resorbed by osteoclasts. Haversian canals of necrotic bone areas were colonized with staphylococcus colonies. Viable areas of compact bone underwent osteoblastic-osteoclastic remodeling, the bone matrix included areas of coarse fibrous bone substance and irregularly located gluing lines. On the periosteal surface of the compact plate, there were volumetric layers of spongy bone substance formed by a finely looped network of massive coarse-fibred trabeculae.

The obtained histomorphological descriptions of the preparations of the operated femur of animals corresponded to the fibrosing with macro- and microabscessing form of chronic osteomyelitis according to V.V. Grigorovsky. A semi-quantitative scoring of the stages of osteomyelitis showed that all animals on the Jupiter scale exceeded the threshold value of 15 points, which indicated an acute course of chronic osteomyelitis. Similarly, the HOES score exceeded the threshold of 6 points defined for active chronic osteomyelitis. In infected animals, leukocytosis was pronounced, the level of C-reactive protein was not significantly higher, but close to the threshold of significance (p=0.06) relative to the reference values (normal). The average values of these indicators are presented in the table.

Table - Quantitative signs of osteomyelitis in rats on the 21st day after surgery, Me (Q1-Q3) Animals Jupiter scale, score HOES scale, score CRP, mg/ml Leukocytes, 109l Experienced Animals
Reference values 27 (27-28)*
1 (0-3) 9 (7-9)*
1 (0-3) 11.6 (10.8-12.0)
10.6 (10.2-11.4) 8.21 (7.95-16.12)*
5.48 (5.13-5.70) Note: * - values are significant relative to the reference (norm) group at p<0.05.

Thus, a complex of clinical, radiological, histomorphological and laboratory signs allows us to assess the condition of the animals as corresponding to chronic osteomyelitis, which allows the method to be used for its intended purpose.

Claims (1)

A method for modeling osteomyelitis of the femur in small laboratory animals, characterized in that a culture of Stafilococcus aureus is intramedullary introduced into the bone, then an implant is introduced intramedullary, a hook-shaped wire is used as an implant, the bone is stabilized by external fixation, by cantilever insertion of the wire and the connection of the wires with self-hardening plastic.

Images