RU2805815C1 - Method of plastic surgery of bone tissue defects using platelet-rich plasma in experiment - Google Patents

Abstract

FIELD: medicine; orthopedics and experimental medicine.

SUBSTANCE: invention can be used for plastic surgery of bone defects in experiments. In the projection of the frontal bones of rabbits, a midline incision is made in the soft tissues to the bone in the projection of the sagittal suture, a chisel is used to collect bone chips in the projection of the planned defect, then using a bur, a blind hole is formed, which is filled with bone chips mixed with platelet-rich plasma in liquid and gel form, obtained from rabbit venous blood, which is obtained by centrifugation and heating at a temperature of 95°C for 4 minutes. The periosteum is placed over the defect, and the wound is sutured in layers. The ratio of liquid plasma and plasma enriched with platelets in the form of a gel is 1:2.

EFFECT: method improves the process of bone tissue regeneration through the use of platelet-rich plasma in the form of a gel and in liquid form together with bone chips.

2 cl, 3 dwg, 3 ex

Description

The invention relates to medicine and can be used in otorhinolaryngology for plastic surgery of the mastoid cavity in patients with chronic purulent otitis media with cholesteatoma after the sanitizing stage of surgical treatment.

Surgical treatment of patients with chronic suppurative otitis media with cholesteatoma is often accompanied by the removal of a large volume of affected temporal bone tissue. Recently, more and more otosurgeons are inclined to the need for a reconstructive stage of the operation - mastoidoplasty. However, there is still no consensus on the optimal plastic material for performing obliteration. The safest technique is the use of autologous materials: bone chips, which are flat bone plates measuring 1.0-2.5 mm, which are obtained when working on bone with a chisel, as well as bone sawdust in the form of fine bone “meal” formed when working with a bur. , which in English-language literature are more often referred to as bone “pate” (bone ).

One of the promising directions is the use of platelet-rich plasma (PRP) as a stimulator of regeneration, obtained from the patient’s venous blood by centrifugation in special tubes. Thanks to the growth factors included in PRP, bone cell proliferation and angiogenesis are stimulated.

The known method is Askar S. M. et al. [Askar SM. Mastoid Reconstruction With Platelet-Rich Plasma and Bone Pate After Canal Wall Down Mastoidectomy: A Preliminary Report / SM. Askar, I.M. Saber, M. Omar. // Ear, Nose & Throat Journal. - 2019. - P.1-5], which involves the use of bone “pate” obtained from the cortical layer of the temporal bone, as well as platelet-rich plasma, for mastoidoplasty. The bone “pate” is placed on the walls of the mastoid cavity, then a PRP clot obtained by centrifuging venous blood for 12 minutes at a speed of 3200 rpm is placed on top of it. The disadvantage of this method is the use of bone “pate”, which does not ensure the regeneration of bone density tissue, since it is small bone particles that are formed when using a bur on the cortical layer.

There is a method by Elbary M. et al. [Elbary M. Platelet-Rich Plasma in Reconstruction of Posterior Meatal Wall after Canal Wall Down Mastoidectomy / M. Elbary, W. Nasr, S. Sorour. // International Archives of Otorhinolaryngology. - No. 22. - 2017. - P.103-107], in which, in addition to bone “pate” and PRP, a titanium mesh 0.1 mm thick, having pores with a diameter of 2.0 mm, is used. The bone “pate” was mixed with PRP in the form of a paste. A titanium mesh is placed over the mastoid cavity, and the posterior wall of the external auditory canal is formed. Bone pate and PRP are placed on top of the titanium mesh. A muperiosteal flap from the postauricular area is placed into the formed mastoid cavity. The disadvantages of this technique are the use of synthetic material - titanium mesh, the need for additional material costs to purchase it, as well as the use of bone “pate”.

The closest to the claimed technical solution in terms of technical essence and achieved technical result is the method of Torres J. et al. [Torres J. Influence of platelet-rich plasma on bone regeneration: A histomorphometric study in rabbit calvaria / J. Torres, I. Tresguerres, F. Tamimi, C. Clemente, E. Niembro, L. Blanco. // The International journal of oral & maxillofacial implants. - No. 22. - 2007. - P.563-568], who described a method for plastic surgery of bone defects in the parietal bones of rabbits. In the parietal bones of rabbits, 2 identical bicortical holes with a diameter of 10 mm are formed. One of them is filled with PRP in liquid form, the other remains empty. The defects are closed layer by layer with periosteum, subcutaneous tissue, and skin with suturing. Rabbits were removed from the experiment after 2, 4, 6 and 8 weeks. The results were assessed according to histological and histomorphometric studies. The disadvantage of this technique is the lack of bone material, which does not allow the technique to be used for large defects of bone tissue, since in this case it will lead to replacement of the defects only with a soft tissue fibrous substrate. In addition, there is a high probability of the graft being displaced from the bone bed due to its liquid consistency. The described method is adopted as a prototype of the invention.

The present invention is based on the task of creating a new method of plastic surgery of bone defects using platelet-rich plasma and bone chips on an experimental rabbit model to improve the process of bone tissue regeneration by increasing its density due to the presence of the bone component, as well as accelerating regeneration due to the presence of growth factors included in PRP.

The method of plastic surgery of bone defects consists in the following: in the projection of the frontal bones of rabbits, a midline incision is made in the soft tissues to the bone in the projection of the sagittal suture, with a chisel, bone chips are taken from the cortical layer of the frontal bone of the rabbit at the site of the planned defect, then the walls of the defect are smoothed using a bur, a non-through hole is formed without exposing the dura mater, filled with bone chips mixed with platelet-rich plasma in liquid and gel form, obtained from the venous blood of a rabbit by centrifugation in test tubes from Plasmolifting (Russia) and heating, then the wound is sutured layer-by-layer.

The method is illustrated with photographs:

Fig. 1 - hole in the frontal bone of a rabbit, filled with bone chips with platelet-rich plasma in liquid and gel form: A - during surgery, B - 1 month after surgery, C - 3 months after surgery, D - 6 months after surgery ;

Fig. 2 - computed tomography of the frontal bone of a rabbit: A - 1 month after surgery, B - 3 months after surgery, C - 6 months after surgery;

Fig. 3 - morphological examination: A - 1 month after surgery, hematoxylin-eosin staining, magnification x 30; B - 3 months after surgery, hematoxylin-eosin staining, magnification x 50, scale - 200 µm; B - 6 months after surgery, hematoxylin-eosin staining, magnification x 50, scale - 200 µm.

The object of the study were male chinchilla rabbits in the amount of 36 individuals weighing from 2.5 to 3.5 kg, which were divided into 3 groups of 12 individuals depending on the timing of removal from the experiment (after 1, 3 and 6 months). The number of animals for the experiment is determined by the need to obtain results associated with the use of parametric tests. Observation of animals for 6 months is the optimal period for an objective assessment of the process of bone tissue regeneration.

The method is carried out as follows. Before the rabbit is put under anesthesia, blood is taken in a volume of 3-5 ml from the marginal vein of the ear into a test tube from Plasmolifting (Russia), which contains sodium heparin at the bottom using “in vivo” technology with a specialized thixotropic gel, which allows the blood to be separated by centrifugation into factions. The tube is centrifuged at 3200 rpm for 5 minutes. When centrifuged, platelet-rich plasma is formed at the top of the tube above the separation gel, while a red blood cell clot remains at the bottom of the tube. To transfer part of the liquid form of platelet-rich plasma into a gel, liquid PRP is taken into a syringe, which is placed in a thermostat at a temperature of 95°C for 4 minutes. Thus, platelet-rich plasma is converted into a dense gel state, which is subsequently placed into the bone defect together with bone chips and liquid PRP.

Before surgery, the fur from the rabbits' frontal surface is shaved off. After premedication (Sol. Atropini Sulfatis 0.05 mg/kg intramuscularly), sedation (Sol. Rometar 4.0-6.0 mg/kg intramuscularly, then after 20 minutes Sol. Zoletili 100 5.0-10.0 mg/ kg intramuscularly), the animal is placed on its stomach and fixed on a special platform located on the operating table, by the front and hind limbs, the head is secured between four clamps.

After processing the surgical field, the tissue in the area of the frontal bone is infiltrated with Sol. Lidocaini 1%, a midline linear incision of the tissues to the bone (5.0-6.0 cm long) is made in the projection of the sagittal suture. The tissues with the periosteum are shifted with a raspatory laterally from the sagittal suture, and a retractor is installed. Bone chips are taken from the cortical layer of the rabbit's frontal bone in the projection of the planned defect using a chisel, which are flat bone plates measuring 1.5-2.5 mm.

The drill forms a blind hole with a diameter of 5.0 mm and a depth of 3.0 mm. The depth of the hole is determined by the thickness of the rabbit's frontal bone, since in this case there is no exposure of the dura mater, the defect is not a through hole, and has bone walls around the entire circumference. The hole is filled with a mixture of bone chips, platelet-rich plasma in liquid form and gel plasma (Fig. 1A). In this case, the ratio of gel plasma and liquid plasma in the mixture is 1:2.

The wound is sutured in layers with interrupted Vicryl 4/0 sutures. Particular attention is paid to bringing together the edges of the periosteum, the displacement of which to its original place contributes to the fixation of plastic materials in the bone bed and subsequent tissue regeneration.

For 7 days, daily dressings are performed to clean the wound.

One bone chip on the rabbits with a small chisel measured approximately 1.0-2.5 mm in length and 1.0 mm in thickness. The holes were 5.0 mm in diameter and 3.0 mm deep. It took 2-4 chips, 0.3 ml of plasma gel and 0.15 ml of liquid plasma to fill one hole.

In humans, the chips can be made larger, since the surface of the skull is large and is not limited to the rabbit's frontal bone.

Animals were removed from the experiment 1, 3 and 6 months after surgery, 12 animals each, by being put under anesthesia followed by an intravenous infusion of potassium chloride. The results of plastic surgery of bone defects were assessed according to visual examination of the area of plastic surgery, computed tomography of the frontal bones, and histological examination (Fig. 1, Fig. 2, Fig. 3).

Example 1.

Extract from the protocol of experiment No. 1. Chinchilla rabbit, male, weight 2.7 kg. Blood was taken in a volume of 3 ml from the marginal vein of the ear into a Plasmolifting tube, the tube was centrifuged for 5 minutes at a speed of 3200 rpm. A syringe with 1 ml of liquid PRP is placed in a thermostat for 4 minutes at a temperature of 95°C. The fur from the frontal surface of the rabbit is shaved. In a sterile operating room after premedication (Sol. Atropini Sulfatis 0.05 mg/kg intramuscularly), sedation (Sol. Rometar 4.0-6.0 mg/kg intramuscularly, then after 20 minutes Sol. Zoletili 100 5.0-10 .0 mg/kg intramuscularly), the animal is placed on its stomach, fixed on the platform.

After processing the surgical field, tissue infiltration in the area of the frontal bone Sol. Lidocaini 1%, a median linear incision of soft tissue up to the bone, 5.0 cm long, was made in the projection of the sagittal suture; the tissue was displaced and fixed with a retractor. Using a chisel, bone chips were collected in the projection of the future defect; the chips were mixed with PRP in the form of a gel and liquid PRP. The drill created a hole with a diameter of 5.0 mm and a depth of 3.0 mm without exposing the dura mater. The hole is filled with plastic material (bone chips + plasma gel + liquid PRP). The edges of the periosteum are brought together. The wound was sutured in layers with interrupted Vicryl 4/0 sutures. The rabbit was kept under normal conditions for 1 month, after which the animal was removed from the experiment.

After 1 month, the defect was clearly visualized (photo 1B); attention was drawn to the uniform filling of the defect with the graft. According to computed tomography, the contours of the defect edges were unclear due to intense bone regeneration (Figure 2A). A morphological study in the defect area revealed a zone of disturbed architecture of the bone tissue of the skull, represented by irregularly shaped beam structures, between which bone marrow cavities were identified. The bone beams in the area of repair of the formed defect were thin, had a tortuous course with a large number of osteocytes, often located in the form of continuous chains. There was also a slight proliferation of connective coarse-fiber or fibro-reticular tissue in the form of islands, often located paratrabecularly (photo 3A).

Example 2.

Extract from the protocol of experiment No. 13. Chinchilla rabbit, male, weight 3.2 kg. Blood was taken in a volume of 3 ml from the marginal vein of the ear into a Plasmolifting tube, the tube was centrifuged for 5 minutes at a speed of 3200 rpm. A syringe with 1 ml of liquid PRP is placed in a thermostat for 4 minutes at a temperature of 95°C. The fur from the frontal surface of the rabbit is shaved. In a sterile operating room after premedication (Sol. Atropini Sulfatis 0.05 mg/kg intramuscularly), sedation (Sol. Rometar 4.0-6.0 mg/kg intramuscularly, then after 20 minutes Sol. Zoletili 100 5.0-10 .0 mg/kg intramuscularly), the animal is placed on its stomach, fixed on the platform.

After processing the surgical field, tissue infiltration in the area of the frontal bone Sol. Lidocaini 1%, a median linear incision of soft tissue up to the bone, 5.0 cm long, was made in the projection of the sagittal suture; the tissue was displaced and fixed with a retractor. Using a chisel, bone chips were collected in the projection of the future defect; the chips were mixed with PRP in the form of a gel and liquid PRP. The drill created a hole with a diameter of 5.0 mm and a depth of 3.0 mm without exposing the dura mater. The hole is filled with plastic material (bone chips + plasma gel + liquid PRP). The edges of the periosteum are brought together. The wound was sutured in layers with interrupted Vicryl 4/0 sutures. The rabbit was kept under normal conditions for 3 months, after which the animal was removed from the experiment.

After 3 months, the defect became less differentiated against the background of the intact frontal bone (Fig. 1B), however, the boundaries between the bone bed and the plastic material were distinguishable, and good integration of the regenerate with the surrounding bone was noted. According to computed tomography data 3 months after surgery, the defect was poorly visible due to filling with regenerate; the structure combined bone and fibrous components (Fig. 2B). A morphological study revealed an increase in fibrotic reactions with the growth of a small volume of reticular tissue in the intertrabecular spaces. Moreover, the number of osteocytes was less than in 1 month of the experiment (Fig. 3B).

Example 3.

Extract from the protocol of experiment No. 25. Chinchilla rabbit, male, weight 3.5 kg. Blood was taken in a volume of 3 ml from the marginal vein of the ear into a Plasmolifting tube, the tube was centrifuged for 5 minutes at a speed of 3200 rpm. A syringe with 1 ml of liquid PRP is placed in a thermostat for 4 minutes at a temperature of 95°C. The fur from the frontal surface of the rabbit is shaved. In a sterile operating room after premedication (Sol. Atropini Sulfatis 0.05 mg/kg intramuscularly), sedation (Sol. Rometar 4.0-6.0 mg/kg intramuscularly, then after 20 minutes Sol. Zoletili 100 5.0-10 .0 mg/kg intramuscularly), the animal is placed on its stomach, fixed on the platform.

After processing the surgical field, tissue infiltration in the area of the frontal bone Sol. Lidocaini 1%, a median linear incision of soft tissue up to the bone, 5.0 cm long, was made in the projection of the sagittal suture; the tissue was displaced and fixed with a retractor. Using a chisel, bone chips were collected in the projection of the future defect; the chips were mixed with PRP in the form of a gel and liquid PRP. The drill created a hole with a diameter of 5.0 mm and a depth of 3.0 mm without exposing the dura mater. The hole is filled with plastic material (bone chips + plasma gel + liquid PRP). The edges of the periosteum are brought together. The wound was sutured in layers with interrupted Vicryl 4/0 sutures. The rabbit was kept under normal conditions for 6 months, after which the animal was removed from the experiment.

The technical result of the method of plastic surgery of bone defects is confirmed by the results of a visual examination of the area of plastic surgery, computed tomography and morphological examination.

After 6 months, the defect was not visualized (Fig. 1D), it acquired the appearance of intact bone, and complete fusion of the plastic material with the bone bed along the entire perimeter was observed. According to computed tomography, the defect had a heterogeneous structure due to a combination of bone and fibrous regenerate with a predominance of bone, which caused a high average density of the regenerate in Hounsfield units (Fig. 2B).

During a morphological study, pronounced phenomena of bone tissue remodeling with expansion of bone beams were noted in the area of the formed defect (Fig. 3B). There was also a decrease in the bone marrow cavities, in which the severity of fibrotic reactions decreased and the volume of fibrous tissue decreased. There was an increase in bone thickness in the area of the formed defect.

According to the results of the analysis of the average bone density in Hounsfield units according to CT data, 6 months after surgery, the holes filled with bone chips, PRP in liquid form and plasmogel acquired a bone density equal to the density of intact bone tissue (643.3 ± 19.6 HU in projection defects at 652.5 ± 11.2 HU in the area of the intact frontal bone of rabbits).

The method is characterized by atraumaticity, ensures the survival of 100% of animals, which makes it possible to dynamically monitor the process of bone regeneration for a long time. Due to the localization of defects in the frontal bones of rabbits, the likelihood of infection of the plastic area and displacement of plastic material from the bone bed is reduced.

The method allows for safe plastic surgery of bone defects, since only autologous tissues (venous blood and bone chips) are used, which eliminates the possibility of allergic reactions.

The use of plasma gel as a binding component for bone chips and platelet-rich plasma in liquid form technically makes it easier to fill the cavity with plastic material and prevents its further displacement.

Plastic surgery with bone chips together with PRP containing various growth factors allows for complete obliteration of the cavity in the shortest possible time, which reduces the likelihood of recurrence of inflammatory processes.

Claims (2)

1. A method for plastic surgery of bone tissue defects using platelet-rich plasma in an experiment, including the formation of a bone defect in the frontal bone of a rabbit, its obliteration with platelet-rich plasma in liquid form, characterized in that a non-through hole is formed in the frontal bone of the rabbit, and bone chips are collected into projections of the defect, and after the formation of the defect, the hole is filled with bone chips, platelet-rich plasma, and platelet-rich plasma in the form of a gel, which is obtained by centrifugation and heating at a temperature of 95°C for 4 minutes; The periosteum is placed over the defect, and the wound is sutured in layers. 2. The method according to claim 1, characterized in that the ratio of liquid plasma and plasma enriched with platelets in the form of a gel is 1:2.