The Role of Botulinum Neurotoxin A in the Conservative Treatment of Fractures: An Experimental Study on Rats

This paper explores the role of botulinum neurotoxin in aiding fracture recovery through temporary muscle paralysis. Specifically, it investigates the effects of botulinum neurotoxin-induced paralysis of the sternocleidomastoid muscle on clavicle fractures in rats. The research aims to assess safety, effectiveness, and the impact on fracture healing. Healthy male Albino Wistar rats were divided into four groups: clavicle fracture, botulinum neurotoxin injection, both, and control. Surgeries were conducted under anaesthesia, and postoperatively, animals were monitored for 28 days. Euthanasia and radiological assessment followed, examining fracture healing and muscle changes, while tissues were histopathologically evaluated. The modified Lane–Sandhu scoring system was used for the radiographic evaluation of clavicle fractures, and the results varied from complete healing to nonunion. Histopathological examination at 28 days postfracture showed fibrous tissue, mesenchymal cells, and primary callus formation in all groups. Despite varied callus compositions, botulinum neurotoxin administration did not affect clavicle healing, as evidenced by similar scores to the control group. Several studies have explored botulinum neurotoxin applications in fracture recovery. Research suggests its potential to enhance functional recovery in certain types of fractures. Theoretical benefits include managing muscle spasticity, aiding reduction techniques, and preventing nonunion. However, botulinum neurotoxin's transient effect and nonuniversal applications should be considered. The present study found that botulinum toxin had no clear superiority in healing compared to controls, while histological evaluation showed potential adverse effects on muscle tissue. Further research is essential to understand its risk-benefit balance and long-term effects.


Introduction
Te management of fractures and the subsequent recovery process are evolving areas in the feld of orthopedics.A bone fracture is defned as any break in bone continuity, and it is followed by a sequence of countermeasures to achieve healing through the formation of a bony callus [1,2].Te stability of fracture parts is of major importance for callus formation, with unstable fractures posing the risk of nonunion or delayed union and the formation of pseudoarthrosis [1].
Te traditional approach to fracture treatment focuses primarily on reduction via closed manipulation or open (surgical) fxation, followed by immobilization and physical therapy [2,3].A primary principle regardless of the open or closed approach is the maintenance of stability [3].A key role in this is played by the action of the muscles around the fracture, which can displace it [4].In conservative treatment, the need to reduce muscle action on the fracture site is greater as there are no osteosynthesis materials to maintain the reduced position [4].Tis is primarily achieved by immobilization and requires the patients' compliance and eforts to limit movement [2][3][4].However, research has experimented with innovative strategies to enhance healing, and recent advancements have introduced the use of botulinum neurotoxin [5,6].
Botulinum neurotoxin (BoNT), commonly known as Botox, is a potent neurotoxic protein produced by the bacterium of the genus Clostridium [7].Its main action is to prevent the release of the neurotransmitter acetylcholine from the nerve terminals at the neuromuscular junction, by selectively binding to the presynaptic membrane of motor neurons [7].Tis inhibition interrupts the transmission of nerve impulses to the muscle, resulting in paralysis [6,7].Tis paralysis is temporary, and its duration depends on factors such as the type of toxin, dose, recipient organism, route of administration, and the type of nerve terminal [7].Moreover, the efect is localized; thus, muscle relaxation/ paralysis can be targeted [7].
Adverse efects associated with the use of BoNTs are normally limited to the injection site and include pain and irritation [7,8].Nevertheless, serious complications such as iatrogenic botulism have also been reported [7].Tere are seven categories of botulinum neurotoxins known today, A-G, classifed according to their characteristics [7].BoNT A1 is considered the most efective as well as the safest one, which is why it is the most utilized in medicine [7].
Due to the above qualities, Botox has gained immense popularity for its cosmetic applications; however, its therapeutic potential extends beyond aesthetics and has found utility in various medical felds, including orthopedics.Experimental studies on rats have investigated the efect of botulinum toxin injected into the quadriceps muscle on the healing of femur fractures [8].In addition, the action of botulinum toxin has been studied in cases of ectopic ossifcation following fractures, showing promising results [9].
Tis paper explores the applications of botulinum neurotoxins in temporarily paralyzing muscles to aid in fracture recovery and healing.Specifcally, it examines the efects of botulinum neurotoxin A-induced paralysis of the sternocleidomastoid (STM) muscle on the healing of clavicle fractures in Wistar rats.
Clavicle fractures account for 2.6-10% of all adult fractures in the general population, while they are the most common fractures in the paediatric population [10,11].Tey are frequent in athletes, with having the third longest recovery time and return to the feld posttraumatically, with 20% never returning [11].In the general population, conservative treatment seems to be preferable to surgery, while only 55% of athletes receive conservative treatment [10].Fractures mostly occur in the middle third of the clavicle, which is the thinnest and least protected part of the bone due to the absence of ligaments [10,11].Te muscle acting on this centric part of the clavicle is the STM, which constitutes a destabilizer by displacing the bone upwards and backwards [11].Te STM plays a leading role in head and neck movements [12,13].In humans, it arises from the handle of the sternum and the middle third of the clavicle with the two heads forming a common muscle in the mastoid process and upper cervical line [12].Te STM by its action contributes to posture, turning, tilting, and extension of the head, while its action on the sternum and clavicle provides support to the temporomandibular joint during mastication [12,13].It is innervated by the cervical plexus and the accessory nerve (11 th cranial nerve), and its dysfunction can result in different degrees of occipital craniocervical pain, as well as cervical dystonia [12][13][14].Te injection of BoNT into the STM for the treatment of torticollis is steadily gaining ground with many authors recommending it [15][16][17].Similarly, the applications of toxins outside the area of aesthetics include the treatment of overactive bladder [18][19][20].
Tis study is the second part of the experimental series and follows the neurophysiological/electrophysiological study conducted on Wistar rats to determine the safe dose of toxin that completely paralyzes the STM muscle for a minimum of 28 days.Tis frst experiment established that 4 international units (IU) were nontoxic (following the rule of 2 IU/100 g of body weight) and sufcient for the desired paralysis [21,22].In the second arm of the experimental protocol, 4 IU of the toxin was injected intramuscularly following a surgically induced clavicle fracture under direct vision.Te efects of the toxin were tested against a control group injected with normal saline and a group that did not receive any additional treatment following the fracture.Additionally, a group receiving 4 IU of toxin only was included, to evaluate the efects on the muscle tissue independently of the presence of a fracture.Fracture healing and callus formation were examined by radiographic and later histological review.Te aim was to determine whether the paralysis of the muscle acting on the fracture site had any efects on the healing process.A secondary aim of the experiment was to review the efects of the toxin on the muscle tissues at a histological level and determine whether any adversities occurred as a result of the toxin administration.Tis research can provide insight into the expanding application of BoNT in clinical practice, outside the scope of aesthetics, and review the safety and efectiveness of its use in orthopedics.

Animals and Treatments.
Tis is a Level I experimental study, that was conducted on healthy adult male rats of the Albino Wistar breed, between 4 and 6 months of age, weighing between 350 and 450 grams.Rats were chosen because of the similarities of their anatomical confguration to that of humans [23].Studies on the possible efect of gender on the efectiveness of botulinum toxin A muscle injection in rats are lacking; however, studies in humans revealed no diference in the dose and efectiveness of botulinum toxin A muscle injection between males and 2 Te Scientifc World Journal females [24].To our knowledge, the majority of studies that examine muscle integrity and function after botulinum toxin A injection in rodents use male rats, which is the reason only male rats were included in the study [24].Te animals were divided into four groups of seven animals each (a total of 28 animals) and were marked accordingly for recognizing purposes.In Group A, a clavicle fracture was intraoperatively induced.In Group B, 4 IU BoNT was injected intramuscularly into the STM muscle.Group C underwent an intraoperative clavicle fracture and intramuscular injection of 4 IU BoNT into the corresponding STM muscle.Group D received an intramuscular injection of 0.9% normal saline (NaCl) in addition to the intraoperatively induced clavicle fracture.Te groups are given in Table 1.
For the duration of the experiment, the animals were bred (accreditation number EL-54BIObre-43) and kept at the animal facility of the Experimental and Research Centre of Papageorgiou General Hospital Tessaloniki (accreditation number EL-54-BIOexp-01) and consumed food and water ad libitum.Animals were group-housed under fully controlled conditions, and a light/dark cycle of 12 hours was followed.
Te procedures were in full accordance with the European Community Council directive 86/609/EEC.Granting of a research protocol license by the Region of Central Macedonia, General Directorate of Agricultural Economy and Veterinary Medicine, Directorate of Veterinary Medicine, Department of Animal Health and Veterinary Perception, Medicines and Applications after applying for protocol authorization to the Directorate of Veterinary Medicine, Region of Central Macedonia and to the Protocol Evaluation Committee of the General Hospital Papageorgiou Research Center preceded the experiments.Te research protocol experiment permit had the protocol number 497690/12/09/2019(2085). Finally, the instructions of the committee for the use and care of laboratory animals of the Aristotle University of Tessaloniki were followed.
In rats, the STM is composed of two bellies, a superfcial sternomastoid medially and a deeper cleidomastoid laterally [25].Te surgeries were performed under general anaesthesia in aseptic conditions, by one surgeon.A mixture of ketamine/xylazine, 50, and 5 mg/kg was administered to induce general anaesthesia and was followed by cleaning and shaving the skin above the right STM muscle and the right clavicle.An approximately 1.5 cm incision was then made on the anterior surface of the clavicle.
Dissection of the STM muscle and the middle third of the clavicle were performed, protecting the surrounding soft tissues (nerves, vessels).A fracture was induced in the middle third of the clavicle with a straight sharp surgical scissor.Te diameter of the clavicle in the middle third is ≈1.5 mm, which easily allows the fracture to be performed in this way.Animals in groups B and C received 4 IU botulinum toxin and were injected intramuscularly into the corresponding sternocleidomastoid muscle (with BD Micro-Fine + insulin syringes with an integrated, nondetachable needle).Botox type A toxin (BOTOX PD.INJ SOL 100U/VIAL BT X 1VIAL) was used, purchased from Allergan Pharmaceuticals Ltd, a substance approved by the National Medicines Agency (approved 24802.01.01).Te skin was sutured with Nylon 3/0 ETHILON simple interrupted suture.
Postoperatively, all animals were examined daily by a trained veterinary professional for pain, shortness of breath, or discomfort.No abnormal fndings were observed during this period, including unaltered feeding habits and behavior.However, paracetamol (DEPON, BristolMyersSquibb SA), at a dose of 1800 mg/kg/24 h, was administered for 3 days via their drinking water for the elimination of any nondetected postoperative pain.During the postoperative period, the animals were allowed to freely move in their cages and no immobilization of the treated forelimb occurred.Tis was due to the animals' need for the limb for feeding, which would have made obstructing its use unsafe.
On the 28th postoperative day, the animals were euthanized by carbon dioxide (CO 2 ) inhalation, a method that causes minimal psychological stress and does not induce pain [26,27].A radiological review followed euthanasia at the radiological laboratory of the Faculty of Veterinary Medicine of the University of Tessaloniki to study the progress of fracture healing.
After the completion of the radiological control, the clavicles of the animals were dissected at the Experimental and Research Center of General Hospital Papageorgiou in Tessaloniki.Tey were sent to the Laboratory of Pathology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Tessaloniki, for histopathological assessment of clavicle fracture healing and of potential changes in the adjacent STM muscle fber bundles, due to injection with BoNT.

Tissue Preparation for Histopathological Assessment.
Clavicles and STM muscle samples from all animals were fxed in 10% bufered formalin.Sequentially, clavicles were decalcifed in hydrochloric acid and formalin-based solution (OSTEOFAST 1, Biognost, Zagreb, Croatia), washed in tap water and immersed in formalin.All tissue segments were routinely processed, embedded in parafn wax, and 4-6 μm thick sections were stained with hematoxylin and eosin.Blind histopathological evaluation was conducted by a single pathologist.Clavicular fracture healing/repair included scoring according to Huo et al., and the grading scale ranged from 1 to 10 [28].

Statistical Analysis.
All data were analyzed using SPSS 28.0 statistical software.One-way analysis of variance (ANOVA), followed by Tukey's post-hoc analysis, and Te Scientifc World Journal independent samples T-test were used for comparisons between the groups.Homogeneity of variances was tested using Levene's test, and when violated, the nonparametric two-tailed Kruskal-Wallis test was used for multiple comparisons, followed by the Mann-Whitney U test for twoby-two comparisons.All results are expressed as the mean ± S.E.M. Signifcance was set at P < 0.05.

Radiographic Assessment.
Te modifed Lane-Sandhu scoring system was used for the radiographic assessment of clavicle fracture healing in animals treated with the toxin, in those injected with saline and in the group that did not receive any additional intervention (Figures 1-4) [29,36].Animal scores on radiological evaluation ranged from 0 (nonunion) to 10 (complete healing) and are described in detail in Figure 5. .In all experimental animals, regardless of botulinum toxin administration, bridging of the two bone ends was not complete.In addition, histopathological evaluation of the muscle tissue from groups with or without BoNT revealed marked diferences, namely, necrosis and atrophy in the former group (Figures 8 and 9).Following the scoring system of Huo et al. [28], the fractured clavicular area had a mean score of 7.18 ± 0.296 for the control animals and a mean score of 7.17 ± 0.386 for the animals that received a botulinum toxin injection, revealing that botulinum toxin administration on the STM muscle did not afect the healing process of fractured clavicles.

Discussion
Several studies have explored the use of botulinum neurotoxin in fracture recovery and healing.A study by Tukel et al. tested the efect of botulinum toxin type A on mandibular fracture healing in rabbits and demonstrated that BoNT injection into the masseter muscle improved bone healing [5].
Another study investigated the efects of botulinum neurotoxin in ankle fractures associated with Achilles tendon tightness [30].Te results indicated that BoNT injections reduced tendon tension, improved fracture alignment, and facilitated early weight-bearing, thereby accelerating the recovery process [30].
Research and literature aim to investigate other uses of Botox by extending its applications in fracture recovery, facilitation of reduction techniques, and prevention of nonunion [6,9,30].Te theoretical, potential beneft would be that in cases where fractures occur in proximity to muscles afected by spasticity, botulinum neurotoxin injections can help manage the associated muscle tightness and spasms.By relaxing the overactive muscles, Botox could promote fracture alignment and reduce the risk of displacement during the healing process [30].
In addition, complex fractures often require meticulous reduction techniques for proper alignment.BoNT-induced muscle paralysis could facilitate these reduction manoeuvres, making them more manageable for the surgeon.In this way, by minimising muscle tension, BoNT could aid in achieving accurate and stable fracture reduction.
Further application of BoNT could include prevention of delayed or nonunion, a potential complication in fracture healing [1].Excessive muscular forces exerted on fracture sites can hinder the formation of a solid union.By temporarily paralyzing the surrounding muscles, Botox could reduce the mechanical stress on the fracture site, optimising the conditions for bone healing and increasing the chances of successful union [6].
When discussing the above potential applications of the neurotoxins, several factors should be considered.One such consideration is the transient nature of its efect [7].Botulinum neurotoxin-induced muscle paralysis is temporary and reversible, with the efects typically lasting for a few months, depending on the dose and recipient organism before muscle function gradually returns [7,20,24].Terefore, additional interventions, such as physical therapy and rehabilitation, are essential to optimise functional recovery once the paralysis wears of [17].
Moreover, it is important to note that the toxin's applications are not universal [7].Te use of Botox in fracture recovery should be carefully considered, and patient selection should be individualised based on factors such as fracture type, location, associated muscle pathology, and overall patient health [7].A comprehensive evaluation by a skilled specialist is crucial to determine the appropriate application of botulinum neurotoxins in each case [7,20].
Tis experimental study focused on the use of botulinum toxin in the conservative management of clavicular fractures in Wistar rats, tested against a control (NaCl).Points of interest were the physiological response to the injection, Xray changes in the healing process, and histopathological fndings observed in the treated tissues (muscle and bone).
Te amount of toxin that was used and the duration of resulting paralysis were determined by a previous experiment, which established through electromyographic testing that 4 IU of botulinum toxin A, administered by intramuscular injection, resulted in paralysis for at least 28 days, without evident toxicity [22].Tis second part of the experimental series completes this original research into the efects of BoNT-induced paralysis on clavicle fracture healing.

4
Te Scientifc World Journal Radiological evaluation of fracture healing according to the modifed Lane-Sandhu scoring system indicated no clear superiority of the toxin in the fracture healing process when compared to controls and no intervention groups.It is noteworthy that in groups A (fracture only) and D (fracture plus saline), there was one incidence of nonhealing, respectively.Tese isolated fndings are not sufcient to prove any causal relationship between the toxin and the degree of healing achieved but are worth exploring further.Overall, all three fracture groups showed variable results, mostly of average healing, with one animal in each group achieving complete healing.Te histological scoring system by Huo et al. was used for conducting a histopathological evaluation of the healing process [28].Tis showed no signifcant diferences between the experimental and control groups, suggesting that the toxin's administration on the sternocleidomastoid muscle did not afect the healing process of clavicle fractures.
Of interest were other fndings of the histological evaluation of the muscle tissue adjacent to the fractured bone between the experimental and control groups.Lesions observed in STM muscles injected with the toxin were more pronounced than in the other groups.Overall, tissue injected with botulin showed more enhanced cell growth and hypertrophy, also necrosis and atrophy, as opposed to the more subtle changes observed in untreated tissue or tissue injected with saline.
Tese fndings are important to acknowledge as potential limitations or even adverse consequences secondary to botulinum neurotoxin A application.Literature supports the idea that such efects can be long-term and potentially not reversible, even after the toxin's paralytic efect subsides [31].A study on healthy volunteers reviewing treated with Botox against saline controls through magnetic resonance imaging, histochemistry, and electron microscopy indicated that muscle changes were sustained over twelve months and included neurogenic atrophy and degenerative changes of the neuromuscular junction [32].A diferent study, conducted on animal subjects, indicated that repeated administration of the toxin on muscle groups resulted in decreased muscle torque compared to muscles treated only once [31].
Postinjection manifestations reviewed in therapeutic applications of the BoNT in children treated for cerebral palsy showed muscle volume loss, sustained for at least six months [33].At the same time, other research has shown that not only does paralysis not aid fracture healing, but it also interferes with it by causing apoptosis and bone degradation [8,34,35].Although the number of animals used was small, this was a decision made on the balance of ethics aiming to keep their numbers to a minimum, while also producing meaningful results.In addition, the inability to achieve compliance with immobilization in animals is an important limitation to consider when reviewing the results of callus formation and evidence of histopathological healing of the fracture.Imaging was not obtained during the healing period but was instead only completed at the end of the treatment period on day 28, as complete healing is expected to have occurred on healthy rats by this date [37].Although more frequent imaging of the gradual healing process could have provided additional data, this decision was based on the risk versus beneft of putting the animals through general anaesthesia, something which could in turn result in deaths.Finally, fracture union and healing would have been better visualized by additional imaging, such as computed tomography [38].Tis can be considered in future research.
While the applications of botulinum neurotoxins continue to primarily concern aesthetics, therapeutic functions of the toxin are expanding.With this in mind, more research is required to investigate the risks and benefts of the toxin's application in this context, as well as the long-term health sequelae.
Fracture Healing and of the Sternocleidomastoid Muscle.Histopathological evaluation of the fractured clavicles, at 28 days postfracture, revealed the presence of fbrous tissue, mesenchymal cells, osteoblasts, and chondroblasts in varying proportions (Figures6(a) and 7(a)).Te presence of woven bone mixed with cartilage (primary callus) was also observed.Te primary callus consisted mainly of collagen fbres of irregular arrangement and was dominated by preosteoblasts and numerous large osteoblasts (Figures6(b) and 7(b)).Only a small number of osteocytes were found, and calcium deposition was either absent or occurred to a small extent.Te formation of the mature lamellar bone (secondary callus) was seen in only a few of the examined clavicles and consisted mainly of compact and less frequent trabecular bone (Figures6(c) and 7(c))

Figure 8 :
Figure 8: Muscle tissue sections from animal subjects in group B (4 IU toxin only).Images on the (a-c) show diferent degrees of healing according to the Huo scoring system.Hematoxylin and eosin, original magnifcation ×10.(a) Oedema of inter-and endomysium, myofber degeneration, and atrophy characterised by muscle fbers showing rarefaction of myofbrils, size variation, and/or angular shape.Certain fbers are enlarged due to hypertrophy.(b) Oedema of inter-and endomysium appears less intense than in Figure 9. Additionally to atrophy, hypertrophic muscle fbers sometimes with longitudinal subdivision/splitting under the same endomysium are observed.(c) Hemorrhage of the perimysium and presence of myofbers with segmental necrosis (middle right).Necrotic fbers show intense eosinophilic sarcoplasm and absence of striation and pycnotic nuclei.

Figure 9 :
Figure 9: Muscle tissue sections from animal subjects in group D (0.9% NaCl only).Images on the (a-c) show diferent degrees of healing according to the Huo scoring system.Hematoxylin and eosin, original magnifcation ×10.(a) Endomysial oedema and multiple fbers showing myofbrillar rarefaction are noticed.Several myofbers present with intense eosinophilic and homogenous sarcoplasm, small size, and angular shape (atrophy).Perimysium and to a lesser extent endomysium are considerably prominent due to probable fbrosis and the absence and/or shrinkage of fbers.(b) Endomysium shows oedema and several fbers with atrophy and hypertrophy.Characteristic is the presence of a group of muscle fbers, with angular to irregular shape, intensely eosinophilic sarcoplasm, usually small in size and nuclei with pyknosis or karyorrhexis (necrosis) and hypercontraction of certain myofbers.Te latter group presents a hemorrhage at its periphery.(c) A considerable number of fbers present mild depletion of myofbrils and loss of transverse striation.Note an extensive area where segments of adjacent muscle fbers are highly vacuolated, but the sarcolemma appears almost intact.In these regions, sarcoplasm is totally absent (lower right).

Table 1 :
Group allocation of animals according to intervention.