Cholinergic stimulation of fracture healing: An animal study

PS John; Ann Maria John; Varghese Thomas; Yogesh Bharat Dalvi; Ruby Varghese

doi:10.4103/joasis.joasis_21_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 19 | Issue : 1 | Page : 24-28

Cholinergic stimulation of fracture healing: An animal study

PS John¹, Ann Maria John¹, Varghese Thomas¹, Yogesh Bharat Dalvi², Ruby Varghese³
¹ Department of Orthopaedics, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala, India
² Pushpagiri Research Centre, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala, India
³ Pushpagiri Research Centre, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla, Kerala; Department of Chemistry, School of Sciences, Jain Deemed to be University, Bengaluru, Karnataka, India

Date of Submission	19-Sep-2021
Date of Acceptance	19-Sep-2021
Date of Web Publication	26-Jun-2022

Correspondence Address:
P S John
Department of Orthopaedics, Pushpagiri Institute of Medical Sciences and Research Centre, Tiruvalla - 689 101, Kerala
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/joasis.joasis_21_21

Abstract

Background: There have been countless attempts not only to address the problems of a failed union but also to accelerate fracture healing. A review of the literature over the last several decades shows that the various strategies for accelerating fracture healing have not been highly successful. Aims and objectives: Recent discoveries on the positive role of cholinergic activity on bone have been successfully used to investigate new promising therapies for various bone diseases and acceleration of fracture healing based on cholinergic stimulation. Citicoline is a precursor of acetylcholine biosynthesis and can increase the level of acetylcholine and thereby increase cholinergic activity. Hence we have done a study to find out the role of citicoline in accelerating fracture healing. Materials and methods: In this animal experiment model, we have studied the healing of leg fracture in a group of ten rats treated with citicoline injection intraperitoneally and in another group without citicoline supplementation. Results: The citicoline group showed early fracture healing with exuberant callus and the tissues studied from the fracture site showed more thymidine incorporation. Conclusions: This animal study has shown that Citicoline can accelerate fracture healing by cholinergic stimulation.

Keywords: Acceleration, acetylcholine, callus, citicoline, fracture healing

How to cite this article:
John P S, John AM, Thomas V, Dalvi YB, Varghese R. Cholinergic stimulation of fracture healing: An animal study. J Orthop Assoc South Indian States 2022;19:24-8

How to cite this URL:
John P S, John AM, Thomas V, Dalvi YB, Varghese R. Cholinergic stimulation of fracture healing: An animal study. J Orthop Assoc South Indian States [serial online] 2022 [cited 2023 Apr 1];19:24-8. Available from: https://www.joasis.org/text.asp?2022/19/1/24/348311

Introduction

Fracture healing is a complex biological process involving many cellular, molecular, and signaling mechanisms, resulting in regeneration of the bone to restore normal function and morphology.^[1] Any alteration in any of these processes in the biological cascade of fracture healing ends up in the various grades of failure ranging from delayed union to nonunion.^[2] There have been countless attempts not only to address the problems of the failed union but also to accelerate fracture healing.^{[3],[4],[5],[6],[7],[8]} A review of the literature over the last several decades shows that the various strategies for accelerating fracture healing have not been highly successful.^{[8],[9],[10],[11],[12],[13],[14],[15],[16],[17]} Some of the medications used for the management of osteoporosis have shown promising results in the healing of osteoporotic fractures. The role of these medications in the management of nonosteoporotic fractures has not been established.

Recent discoveries on the positive role of cholinergic activity on bone have been successfully used to investigate new promising therapies for various bone diseases and acceleration of fracture healing based on cholinergic stimulation.^{[18],[19],[20],[21],[22],[23],[24]}

As we all know, bone remodeling involves bone resorption by osteoclasts and subsequent formation of new bone by osteoblasts. This process regulates the biomechanical features of bone and maintains the homeostasis of essential ions in the body. Alteration in the bone remodeling process results in diseases of bone and failure in fracture healing.^[25] Bone remodeling is regulated by the two branches of the autonomic nervous system; the adrenergic and cholinergic branches. Adrenergic activity favors bone loss, whereas cholinergic activity has been recently shown to increase bone mass. The cholinergic system regulates the body's metabolic activity through the use of acetylcholine (ACh) as a signal transmitter. ACh is biosynthesized by choline acetyltransferase, and it is stored in small synaptic vesicles through the action of the vesicular ACh transporter enzyme, to be released via exocytosis into the synapse space.^[26] The secreted ACh targets either nicotinic or muscarinic receptors. Nicotinic receptors are classified into five subtypes including alfa, beta, gamma, delta, and theta which assemble to form ionic channels. Muscarinic receptors are guanine nucleotide protein-coupled receptors and are composed of five subunits; m1, m2, m3, m4, and m5. ACh signal is terminated by its degradation through the enzyme ACh sterase (AChE).^[25]

The available literature suggests that the inhibition of cholinergic activity at the bone level and in the central nervous system reduces bone mass and boosting of ACh activity might have an anabolic effect on bone formation.^[10],[11]

One way of stimulating cholinergic receptors is by the administration of cholinergic agonists such as ACh esterase inhibitors (AChEIs). AChEIs are a group of drugs that cause stimulation of cholinergic receptors by inhibiting the action of AChE and increasing the levels of ACh in the synaptic space.

A recent clinical study by Tamimi et al. has reported that treatment with centrally acting AChEI that stimulates both nicotinic and muscarinic receptors such as donepezil and rivastigmine was associated with a lower risk of hip fracture in Alzheimer's disease patients.^[27] In another very interesting study, Tamimi et al. have again concluded that in elderly patients with Alzheimer's disease, the use of AChEI might be associated with an enhanced fracture healing and minimal complications. If AChEIs can accelerate fracture healing by increasing the level of ACh, it is logical to assume that any pharmacological agent that increases the ACh level, should be able to accelerate fracture healing by the same mechanism. Citicoline is a precursor of ACh biosynthesis and can increase the level of ACh.^[25],[26] Hence, we hypothesized that the intraperitoneal administration of citicoline will accelerate the fracture healing.

Materials and Methods

In this background, to test our hypothesis, we have done an experimental study in the department of orthopedics in association with the Research Department of Pushpagiri Institute of Medical Sciences, Thiruvalla. Eight-week-old male Sprague − Dawley inbred rats (150–250 g) were purchased from Small Animal Breeding Section, Kerala Veterinary and Animal Sciences University Mannuthy, Kerala, India. Rats were housed in a single polycarbonate cage with small animal wood bedding and allowed to habituate to the animal laboratory conditions for 2 weeks before the commencement of the experiment. All rats were maintained under standard conditions of temperature (25°C ± 2°C) and humidity, with a light/dark cycle of 12 h and free access to standard food (Champaka feeds and foods, Bangalore, India) and ultra violet sterile water. All animal experiments were carried out with the prior Approval of the Institutional Animal Ethics Committee (No. 602/PO/Re/S/2002/CPCSEA) and were conducted strictly adhering to the guidelines of CPCSEA, Animal Welfare Division, Government of India.

Initially, the rats were randomly divided into two groups: Group A (n = 10) and Group B (n = 10). Animals were anesthetized with a single intramuscular cocktail dose of xylazine hydrochloride 13 mg/kg Bodyweight (Indian Immunologicals Limited, Hyderabad, Telangana, India), and ketamine 65 mg/kg Bodyweight (Neon Laboratories Limited, Andheri [East], Mumbai, Maharashtra, India).

The fracture was induced in the left tibia of all the rats under anesthesia. The fractures were splinted and the Group-A rats were given 100 m/kg of citicoline injection intraperitoneally once daily for 1 week. The union of fractures was assessed once every week by observing the gait and with radiographic evaluation. On the 2^nd week when the callus was maximum, a sample of tissue was taken from the fracture site by aspiration. The aspirate was transferred to phosphate-buffered saline medium. The solution was centrifuged and the pellet was taken and spread into an Roswell Park Memorial Institute (RPMI) medium with 10% fetal calf serum. These cells were checked for viability by adding Triphan blue. The cells were then incubated with radioactive Thymidine for 24 h. The medium was then centrifuged and the supernatant drained away. The cells obtained were then resuspended in RPMI medium and kept in a scintillation chamber and the radioactive thymidine incorporation was then measured.

Analysis of gait score

Gait analysis is a helpful tool to comprehend behavioral changes (e.g.,: limping) in preclinical fracture models. In this study, gait analysis was carried out by allotting two independent blindfold observers for subjective clinical assessment. The animals were carefully observed throughout the entire experimental period by the observers and assessed every day till the completion of the experiment.

Results

All the rats developed painful limping immediately after the fracture. The entire citicoline administered group returned to normal gait in 3 weeks. The test group continued to have some abnormal gait for more than 4 weeks. All the X-rays of the fractured leg showed an increased callus formation in the citicoline injected rats compared to the control group [Figure 1]. The results of thymidine incorporation demonstrated that the citicoline group had increased incorporation of radioactive thymidine compared to the control group [Figure 2]. The statistical analysis by Student's t-test showed a P < 0.001 which is significant. On follow-up all the fractures in the citicoline injected, rats united in 3 weeks, whereas all the rats in the control group took more than 4 weeks for fracture healing.

Figure 1: Citicoline rats at 2 weeks control rats at 2 weeks

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Figure 2: Thymidine incorporation assay revealed significantly (P < 0.001) high values in the test group (treated with citicoline) when compared to the control group. It means that a higher cell proliferation potential was observed for the callus harvested from the test group. This suggests a better fracture healing potential for the callus harvested from the test group when compared to the control group

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Radiological examination of fractured legs in control and test groups

Radiometric assay

Discussion

Many medical conditions and therapeutic agents that alter the ACh signaling system have been found to have interesting effects on bone function.^{[1],[2],[3],[4],[5],[6],[7],[8]} At the presynaptic level, botulinum neurotoxin inhibits the release of ACh from motor neurons, and has been found to impair bone healing and induce a decrease in bone mineral content.^[4],[5] However, bone density does not improve after the recovery of muscle function, indicating an effect of botulinum toxin on bone tissue, unrelated to muscle function.^[5] Poliomyelitis destroys motor neurons that use ACh neurotransmitters. Patients suffering from poliomyelitis experience impaired bone growth in the limbs affected by nerve depletion. However, many years after recovery of muscle activity, poliomyelitis patients tend to develop osteoporosis in a much larger proportion than the rest of the population.^[9] At the postsynaptic level, smokers are well known to have^[6],[7],[8] impaired bone healing, in part due to the effect of nicotine. Smokers have high concentrations of nicotine in the blood that interacts with the nicotinic ACh receptor (nAChR), causing desensitization of these receptors. All these observations suggest that ACh and its receptors in bone tissue may play certain roles in the process of bone remodeling.

Recent research has revealed the presence of a number of AChR subunits in bone cells.^[1],[2],[3] nAChR subunits have been detected in human primary bone cells, mesenchymal stem cells (MSCs), and osteoblasts, whereas muscarinic receptors have been detected in MSCs.^[2],[3],[6] AChR and cholinergic components expressed on osteoblasts may play a possible role in regulating alkaline phosphatase activity as well as the proliferation and differentiation of osteoblasts.^[10] Moreover, the enzyme responsible for metabolizing ACh at the AChR site, AChE is highly expressed on bone cells, especially during the osteoblast differentiation stage.^[11],[12] Altogether, the available literature seems to indicate that the inhibition of the AChR seems to cause a reduction in bone turnover,^{[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14]} and boosting of ACh activity would have an anabolic effect on bone formation. One way of stimulating cholinergic receptors is by the administration of cholinergic agonists such as AChEIs. AChEIs are a group of drugs that cause stimulation of cholinergic receptors by inhibiting the action of AChE and increasing the levels of ACh in the synaptic space. A recent clinical study by Tamimi et al. has reported that treatment with centrally acting AChEI that stimulates both nicotinic and muscarinic receptors such as donepezil and rivastigmine was associated with a lower risk of hip fracture in Alzheimer's disease patients. In another very interesting study, Tamimi et al. have again concluded that in elderly patients with Alzheimer's disease, the use of AChEI might be associated with an enhanced fracture healing and minimal complications.^{[27],[28],[29]}

If AChEIs can accelerate fracture healing by increasing the level of ACh, it is logical to assume that any pharmacological agent that increases the ACh level, should be able to accelerate fracture healing by the same mechanism. Citicoline is a precursor of ACh biosynthesis and can increase the level of ACh.^[30] Citicoline is the generic name for synthetic Cytidine Diphosphate Choline (CDP-choline), an organic molecule produced endogenously also and found in all living cells. CDP-choline is also a precursor for the synthesis of phospholipids that are essential constituents of the cell membrane. Because cell membranes have a very high turnover rate,^[31] these phospholipids must be continuously synthesized to ensure the adequate function of cells. Citicoline is a very safe drug considered as a nutraceutical used all over the world for the past so many decades, from 1970, with no major side effects. It has been extensively used in the treatment of various neurodegenerative disorders, head trauma, stroke, brain aging, cerebrovascular pathology, Alzheimer's disease, learning disabilities, and memory loss. It is popularly known as a brain nutrient.^[32]

Conclusions

In this study, leg fracture in rats treated with citicoline (Group 1) demonstrated significantly faster rate of fracture healing compared to the control rats (Group 2). Early callus formation and abundant callus were observed in the treatment group. The cell proliferation was much more in the callus of the treatment group as evidenced by the thymidine incorporation test. Based on the results of this study, we hypothesize that the administration of citicoline may have a beneficial effect on bone turnover and hence the acceleration of fracture healing.

Ethical clearance

animal experiments were carried out with the prior approval of the Institutional Animal Ethics Committee (No.602/PO/Re/S/2002/CPCSEA) and were conducted strictly adhering to the guidelines of CPCSEA, Animal Welfare Division, Government of India.

Acknowledgments

Thanks are due to Rev. Dr. Mathew Mazhavencheril, Research Director, Pushpagiri Groups of Institutions, Tiruvalla, Kerala, India, for his administrative support toward this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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