Effects of gamma radiation and post-operative cisplatin injection on the incorporation of bone allografts in rats

Вплив γ-випромінювання та післяопераційного введення цисплатину на інкорпорацію кісткових алоімплантатів у щурів Вирва О.Є.1, ORCID: 0000-0003-0597-4472, e-mail: dr.olegvyrva@gmail.com Головіна Я.О.1, ORCID: 0000-0002-1605-9109, e-mail: dr.yanina.golovina@gmail.com Ашукіна Н.О.1, ORCID: 0000-0002-0478-7440, e-mail: nataliya.ashukina@gmail.com Малик Р.В.2, ORCID: 0000-0001-9070-4834, e-mail: dr.roman.malyk@gmail.com Данищук З.М.1, ORCID: 0000-0003-2968-3821, e-mail: zinada1962@gmail.com 1Державна установа «Інститут патології хребта та суглобів імені професора М.І. Ситенка Національної академії медичних наук України», Харків, Україна 2Харківська медична академія післядипломної освіти Міністерства охорони здоров’я України України, Харків, Україна

Background. The reconstruction of long bone defects that occur after resection of tumors is a problem that requires constant study. Bone allografts are often used in this scenario. Unfortunately, while they are prepared, allografts partially lose their strength and osteoinductive properties; their survivability in oncological patients is only 40% after 10 years. This is why the search for superior allograft treatment methods and the study of allograft remodeling and incorporation in oncological patients, whose state has been affected by radiation or chemotherapy, is an area of interest. Purpose -study the structure of bone tissue in the distal metaphysis of a rat's femur after bone allograft implantation (sterilized using gamma radiation or antibiotic saturation) and post-operative intraperitoneal cisplatin injection. Materials and Methods. Experiments were performed on 20 male white rats aged 5-6 months at the beginning of the experiment and weighed 365.8 ± 6.4g. All rats underwent a surgery that created a defect in the distal metaphysis of the femur which was filled with a bone allograft treated with gamma radiation (Control-1 and Experimental-1 groups) or saturated with an antibiotic (Control-2 and Experimental-2 groups). 14 days after allograft implantation, animals from the control groups received an intraperitoneal injection of 2.0-2.4 ml of 0.9% NaCl, while animals from the experimental groups received 2.5mg/kg of cisplatin. Histological analysis and histomorphometry were completed 30 days after the surgery. Results. 30 days after the operation, the smallest relative area of bone tissue (11.79%) was observed in rats from the Experimental-1 group, with gamma radiation treated allografts and post-operative intraperitoneal cisplatin injections. A somewhat higher value was found in the Experimental-2 group (antibiotic saturation + cisplatin) -31.64%. In the control groups, (intraperitoneal injection 0.9% NaCl), the relative area of bone tissue was 16.7% (Control-1, gamma radiation treatment) and 58.09% (Control-2, antibiotic saturation). The relative area of fibrous tissue was the largest in the Experimental-1 group -31.55% and the smallest in the Control-2 group -12.79%. Conclusions. Allograft remodeling occurs along with the formation of bone and fibrous tissue when allografts are used to fill defects in the distal femoral metaphysis of rats, However, the relative percentages of those tissues depend on the allograft sterilization method and the use of cytostatic agents. The largest relative percentage of bone tissue (58.09%) was obtained using an allograft saturated with antibiotics and without the administration of cisplatin. The smallest (11.79%), on the other hand, occurred in gamma radiation treated allografts with cisplatin injected intraperitoneally after the operation Реконструкцію післярезекційних дефектів довгих кісток у разі їхнього ураження пухлинами є актуальною проблемою, що потребує постійного вивчення. Протягом декількох десятиріч у світі триває пошук «ідеального» імплантаційного матеріалу для цієї мети. Основними вимогами до імплантатів є їхня біоінертність, можливість замістити дефекти кісток різного розміру та форми, біосумісність із прилеглими тканинами реципієнта, міцність [1,2]. Сьогодні використовують безліч технік для реконструкції великих дефектів кісток і суглобів. Серед них основними є кісткова пластика з використанням ауто-, ало-та ксенотрансплантатів, дистракційний остеосинтез, заміщення дефектів біоматеріалами, модульне та індивідуальне ендопротезування [3][4][5].
The reconstruction of long bone defects that occur after resection of tumors is a problem that requires constant study. For several decades, scientists all over the world are looking for the perfect implant material for this purpose. The main requirements for the bone implant include bioinertness, the ability to replace defects of various sizes and shapes, compatibility with adjacent recipient tissue, and strength [1,2]. Today, many different techniques are used to reconstruct large bone and joint defects. The main ones include filling the defects with auto-, allo-, and xenografts or biomaterials, distraction osteogenesis, and modular and individual endoprosthesis [3][4][5].
Even so, combined methods of reconstructing large segmental bone and joint defects, such as distraction osteogenesis using bone autografts and allograft prosthesis composite (APC), are currently gaining in popularity. This combination of different methodologies makes it possible to minimize the disadvantages of each one and effectively utilize their advantages [6,7].
Allograft reconstruction of bone defects that occur after tumor resection is a common and mostly successful method for surgically treating patients. This method has clear advantages over the rest (biological recovery of the bone tissue and muscle attachment zones). However, the complications rate remains high, which limits the possibility of using allografts in this scenario [8]. The most common complications observed in oncological patients include infections (from 8.3% to 20% [9,10,11]), fractures, pseudoarthrosis (from 8% to 14%), and contractures [12][13][14]. More rarely, allograft resorption occurs, which is caused by a decline in the patient's immunological status after polychemotherapy. As a result of several studies, the average length of time required for union between the allograft and recipient's bone was determined to be approximately 8 months [15,16].
In order to fill large bone defects, bone oncologists usually use structural and particulate (granular, bone chip) allografts that are sterilized using gamma radiation (15-35 kGy) [17], or low temperatures (fresh-frozen, temperatures below -75°С) [18]. It was determined that the use of fresh-frozen allografts increases the risk of infection [18], while sterilizing with gamma radiation increases the risk of allograft fracture. Since radiation affects the structure of the allograft, using large ones is not recommended in APC.
The refinement of treatment methods for bone allografts and study of their interaction with cytostatic drugs is a potential area for further research.
Objective -study the structure of bone tissue in the distal metaphysis of a rat's femur after bone allograft implantation (sterilized using gamma radiation or antibiotic saturation) and post-operative intraperitoneal cisplatin injection.  [20,21].

Animals
The plan for the study was approved by the bioethics committee at the Sytenko Institute of Spine and Joint Pathology (Protocol No. 204 from 15.06.2020) in accordance with the Ukrainian Law «On the Protection of Animals from Brutal Treatment», the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and Directive 2010/63/EU [20,21].
Experiments were performed on 20 male white rats from the population of the experimental biological clinic of the Sytenko Institute of Spine and Joint Pathology. The animals were aged 5-6 months at the beginning of the experiment and weighed 365.8 ± 6.4 g. There were 5 rats in each cage, with surrounding temperatures of 22-24°С and a 12-hour light period, and access to food and water.

Study design
All rats underwent a surgery that created a defect in the distal metaphysis of the femur and were randomly divided into groups depending on the material used to fill the defect and whether cisplatin was used: -Control-1 (n=5) and Experimental-1 (n=5) -allografts (diameter 2 mm, height 3 mm) sterilized with gamma radiation; -Control-2 (n=5) and Experimental-2 (n=5) -allografts (diameter 2 mm, height 3 mm) saturated with Ceftriaxone.
The animals were euthanized by administering a lethal dose of anesthetic (sodium thiopental, 90 mg/kg intramuscularly) 30 days after the surgery.

Preparation of bone allografts
Cancellous bone allografts (diameter 2 mm, height 3 mm) were prepared from the femoral and tibial metaphyses of 5 donor rats using previously defined methods [23]. Allografts were sterilized using gamma radiation (15-25 kGy) or submerged for 24 hours in a 4°C Ceftriaxone solution («Kyivmedpreparat» PJSC in «ARTERIUM LTD», Ukraine) with 0.9% sodium chloride as the solvent. 1g of Ceftriaxone was dissolved per 10 ml of solvent.

Surgery
The operations were completed in aseptic and antiseptic conditions under general anesthesia (ketamine, 50mg/kg intramuscularly). After the fur was shaved off the Histology Femurs that were operated on were taken out, cleaned of soft tissues, and stored in 10% neutral formaldehyde. After 4 days, the bones were placed in 10% formic acid to decalcify and a distal metaphysis with the implantation area was cut out of each bone. The distal metaphyses of the femurs were dehydrated in isopropyl alcohols of increasing concentration and a mixture of paraffin and xylene (1:1), and embedded into paraffin. Frontal histological sections 5-6 µm in width were stained using hematoxylin and eosin (H&E) and Van Gieson's stain, and analyzed under a BX63 light microscope (Olympus, Japan). Digital images were obtained using a DP 73 camera (Olympus).

Histomorphometry
Using the «Cell Sens Dimention 1.8.1» software (Olympus, 2013), the areas of newly formed tissues (bone and fibrous) were measured in two central sections for each animal. After measurement, the percentage of bone tissue (B%) and fibrous tissue (F%) relative to the total area of the defect was calculated.
Lysis formation only occurred around allograft fragments in the Experimental-1 group. In the Control-2 group, cancellous bone tissue of the lamellar structure and periosteum were formed. H&E. A -allograft, F -fibrous tissue, B -newly formed bone, γ-rays -gamma radiation. Images from section B are fragments of images from section A
The fibrous tissue was dense and contained large numbers of fibrocytes with light-colored cytoplasm and hyperchromic nuclei. Newly-formed bone trabeculae were located on the perimeter of the defect, specifically on the border of the host bone. In the Control-1 and Experimental-2 groups, the trabeculae also formed right on the surface of the allograft. Osteocytes with hyperchromic nuclei and cytoplasm of relatively small size were found in the lacunae of the trabeculae. Functionally active osteoblasts were located on the surface of the trabeculae, which is evidence for reparative osteogenesis occurring in those areas.
As a result of the histomorphometry, it was determined that when allografts were sterilized using gamma radiation, B% was lower than F%: by 1.66 times (p=0.004) in the Control-1 group, by 2.8 times (p<0.001) in the Experimental-1 group. On the contrary, in the Experimental-2 group, B% was higher than F% by 1.9 times (p<0.001) (fig.3).
The allografts in the Control-1, Experimental-1 and Experimental-2 groups showed evidence of remodeling. Blood capillaries and undifferentiated cells grew deep into the allograft, where loose fibrous tissue was also observed. Lysis was found around some of the allograft fragments in the Experimental-1 group. Recovery of the cortical layer of the distal femoral metaphysis was not observeddense fibrous tissue was located in that area ( fig. 2).
The structure of the defect differed in the Control-2 group (allograft sterilized through saturation with antibiotic, intraperitoneal injection of 0.9% NaCl) in comparison with the other groups. Specifically, the allograft was almost completely remodeled, with cancellous bone tissue of the lamellar structure and red and yellow bone marrow forming in its place. Newly-formed bone trabeculae formed close to one another and perpendicular to the bone axis. A large number of brightly-colored osteocytes were found inside the trabeculae (fig. 2). As opposed to the other experimental groups, newly-formed bone tissue was also located in the cortical defect and was firmly connected Ukrainian journal of radiology and oncology. 2021;29(3):51-62 ISSN 2708-7166 (Print) ISSN 2708-7174 (Online)
to the host cortex. B% exceeded its counterpart in the Control-1 group by 3.46 times (p<0.001) ( fig. 3). In some locations, fibrous tissue with a low cell density was observed between the newly-formed bone trabeculae. F% was 4.54 times smaller than the B% (p<0,001) and 2.1 times smaller than its counterpart in the Control-1 group (p<0,001). Areas of periosteal and endosteal bone formation were found. A periosteum was formed over the area of the defect Inflammation was not observed in any of the samples. Destructive changes were found in the host bone when cisplastin was used: damaged matrices, formation of fissures, areas without cells, small areas of lysis of bone marrow and trabeculae, and swelling of the bone marrow.

Discussion
Bone allografts are often used to reconstruct large bone defects that occur after tumor resection because the method using which allografts are obtained does not require additional painful surgery and makes it possible to avoid issues with the donor site, as opposed to autografts. Unfortunately, while they are prepared, allografts partially lose their strength and osteoinductive properties; their survivability in oncological patients is only 40% after 10 years [11,24]. This is why the search for superior allograft treatment methods and the study of allograft remodeling and incorporation in oncological patients, whose state has been affected by radiation or chemotherapy, is an area of interest.
This study evaluated the effect of different sterilization methods (gamma radiation or antibiotic saturation) and post-operational injections of cisplatin on the remodeling of allografts. Allografts were placed in the transcortical defects of distal femoral metaphyses in rats; 14 days after the operation, the animals received an intraperitoneal injection of 2.0 -2.4 ml of 0.9% NaCl (Control-1 and Control-2 groups) or 2.5mg/kg of cisplatin (Experimental-1 and Experimental-2 groups).
30 days after the operation, remodeled allografts, parts of which were replaced with bone and fibrous tissue, whose areas varied between groups, were observed in the defects. The worst result, from the perspective of bone tissue formation, (11.79%) was reached in the Experimental-1 group, when allografts were sterilized using gamma radiation and cisplatin was injected intraperitoneally after the operation. This finding can be explained by the influence of two negative factors. On one hand, it is known that sterilizing using gamma radiation leads to a decrease in biomechanical properties and lifespan of bone allografts, which is caused by the destruction of collagen in the bone matrix [17,19,25]. On the other hand, it was found that post-operative injection of cisplatin significantly decreases the resorption ability of the allograft and bone formation [26], and significantly reduces bone formation during distraction osteogenesis [27,28]. It was demonstrated that cisplatin inhibits the proliferation and stimulates the apoptosis of mesenchymal stem cells of the bone marrow, which are one of the sources of bone regeneration [29]. Consequently, the removal of one of those factors made it possible to obtain an increased relative percentage of bone tissue: in the Experimental-2 group (antibiotic saturation + cisplatin) -31.64%. Ukrainian journal of radiology and oncology. 2021;29 (3)
As a result of the conducted study, it was determined that when allografts are used to fill defects in the distal femoral metaphysis of rats, allograft remodeling occurs along with the formation of bone and fibrous tissue. However, the relative percentages of those tissues depend on the allograft sterilization method and the use of cytostatic agents. The largest relative percentage of bone tissue (58.09%) was obtained using an allograft saturated with antibiotics and without the administration of cisplatin. The smallest (11.79%), on the other hand, occurred in gamma radiation treated allografts with cisplatin injected intraperitoneally after the operation.
Nonetheless, that figure is significantly smaller than the value in the Control-1 group (58.09%), where both of the negative factors were absent. This was, as expected, the best result.
The relative percentage of fibrous tissue was highest in the Experimental-1 group -31.55%. This may be caused by an acute inflammation that occurred after the implantation of gamma radiation treated allografts that may have led to allograft lysis and the formation of large amounts of fibrous tissue [30]. The smallest relative percentage of fibrous tissue was in the Control-2 group -12.79%.
Comparing the results of past studies with the ones obtained in this work, it can be concluded that combining gamma radiation treated allografts with the post-operational injection of cytostatic agents (cisplatin) leads to reduced bone regeneration. Also proven was the negative impact of cytostatic agents on the incorporation of the allograft (Experimental-1 group).