The effect of <i>Aloe vera </i>ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study

Thomas Erwin Christian Junus Huwae; Mohamad Hidayat; Hidayat Sujuti; Retty Ratnawati

doi:10.12688/f1000research.110244.1

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Research Article

The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study

[version 1; peer review: 1 approved, 1 not approved]

Thomas Erwin Christian Junus Huwae ¹, Mohamad Hidayat¹, Hidayat Sujuti², Retty Ratnawati³

PUBLISHED 30 Mar 2022

Author details Author details

¹ Department of Orthopedics and Traumatology, Faculty of Medicine Universitas Brawijaya, Saiful Anwar General Hospital, Malang, East java, 65111, Indonesia
² Department of Ophthalmology, Faculty of Medicine Universitas Brawijaya, Malang, East Java, 65111, Indonesia
³ Department of Physiology, Faculty of Medicine Universitas Brawijaya, Malang, East Java, 65111, Indonesia

Thomas Erwin Christian Junus Huwae
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Mohamad Hidayat
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Hidayat Sujuti
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Retty Ratnawati
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Tourniquet is a common instrument used in Orthopedics field to reduce blood loss, providing a better operating field. One of the deleterious effects of tourniquet is ischemic-reperfusion injury, which occurs after deflation of the tourniquet. One of the possible ways to mitigate ischemic-reperfusion injury is by administrating antioxidants to reduce oxidative stress. Natural ingredients like Aloe vera are well known for its antioxidant and anti-inflammatory activities. This experimental study was conducted by fracturing the tibia of male Wistar strain rats (Rattus norvegicus) and administering Aloe vera gel orally as antioxidant.
Method: A total of 18 rats were used in this study, divided into 6 groups. The first group was the control group, that included rats with fractured tibia not receiving torniquet and aloe vera treatment. The second and third groups were rats with fracture, who received torniquet application for one hour and two hours respectively without aloe vera treatment. The rest were the treatment groups, the rats with factures were given different dosages of Aloe vera extract: 40 mg/kg bodyweight (BW), 60 mg/kg BW, and 80 mg/kg BW. Three hours after administration, the tourniquet was inflated for two hours, followed by its deflation. The tibia was harvested to examine levels of superoxide dismutase (SOD), Malondialdehyde (MDA), and Bone morphogenetic protein 7 (BMP-7), and callus diameter, and osteoblast numbers were evaluated.
Result: Administration of Aloe vera extract reduced the oxidative stress parameters (SOD and MDA). Tourniquet application decreased the osteoblast cell count and callus diameter, and administration of Aloe vera extract increased both variables close to the control value (p<0.05).
Conclusion: The result of this study suggests that Aloe vera extract could be used to ameliorate ischemic-reperfusion injury.

Keywords

Aloe vera, fracture healing, ischemic-reperfusion injury, tourniquet, Superoxide dismutase, Malondialdehyde, Bone morphogenetic protein

Corresponding author: Thomas Erwin Christian Junus Huwae

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2022 Huwae TECJ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Huwae TECJ, Hidayat M, Sujuti H and Ratnawati R. The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2022, 11:372 (https://doi.org/10.12688/f1000research.110244.1) First published: 30 Mar 2022, 11:372 (https://doi.org/10.12688/f1000research.110244.1) Latest published: 30 Mar 2022, 11:372 (https://doi.org/10.12688/f1000research.110244.1)

Introduction

Tourniquet is frequently used in orthopedic procedure involving fractures of long bone to achieve a bloodless operation field, or it can also be used in elective procedure such as total knee arthroplasty (TKA).¹^,² The application of tourniquet, however, poses several complications including local neurovascular complication, systemic complication, nerve injury, post-tourniquet syndrome, etc.³ The deleterious effects of the tourniquet is caused by the direct compression and by the ischemic-reperfusion injury occurring after deflation.⁴ A study in Norway found that the incidence of tourniquet-related complications in orthopedic surgery was about 0.032%, more frequently affecting lower extremities compared to upper extremities.⁵

Ischemic-reperfusion injury is also one of possible serious complication of tourniquet application.⁶ Ischemic reperfusion can affect the limb itself (muscle and joint), or it may affect distant organ.⁴ Tourniquet-related ischemia-reperfusion injury can increase the superoxide synthesis and suppress endogenous antioxidant activity. Moreover, application of tourniquet by only 2 hours showed elevated creatine phosphokinase and lactic acid in animal model, suggesting that muscle damage occurs.⁷ On the other hand, the damage to distant organ is thought to be caused by superoxide ions entering circulation through venous blood flow.

There are several strategies suggested to ameliorate the ischemic-reperfusion injury after tourniquet application: ischemic preconditioning and postconditioning, reperfusion interval, and administration of pharmacologic agents.⁸^–¹⁰ Reperfusion interval is proven to reduce the inflammatory markers in the distant organ, in this case the lung.¹⁰ However, the drawback of this method is the prolonged operating time. Thus, the authors would like to explore a measure which could be applied preoperatively to prevent this.

One of possible methods to reduce oxidative stress is by administering exogenous antioxidant, and this may be done preoperatively. Several antioxidants have been used in previous experimental studies: methylene blue, tramadol, magnesium sulphate, platonin, and others.¹¹^–¹⁴ One of naturally occurring antioxidant often used in various settings is Aloe vera. Aloe vera gel has been used to counter ischemic-reperfusion injury in the lung, kidney, and liver.¹⁵^,¹⁶ However, the use of this agent in bone tissue is yet to be studied. Thus, the author aimed to evaluate the antioxidative effect of Aloe vera gel to mitigate ischemic-reperfusion injury after tourniquet application.

Methods

This experimental study was conducted between January–March 2019 at the Faculty of Medicine, Universitas Brawijaya, Indonesia. The acclimatization, maintenance, and intervention of the samples were done in the Animal Model Unit, Laboratory of Pharmacology. The measurement of levels of superoxide dismutase (SOD), Malondialdehyde (MDA) were done in the Physiology Laboratory. The evaluation of Bone morphogenetic protein 7 (BMP-7) level was conducted in the Central Biomedical Laboratory. The callus and osteoblast evaluation were conducted in the Pathology Laboratory. The Ethics Committee of Faculty of Medicine, Universitas Brawijaya approved all animal protocols, and all subsequent experiments with the registration number: No. 72/EC/KEPK-S3/03/2020. All animal protocols, and all subsequent experiments were also carried out according to the ARRIVE guidelines and regulations.

Animal model

The animal models in this study were 18 male rats (Rattus norvegicus), divided into 6 groups with 3 rats in each group. Male rats were used because the use of male rats is already established in fracture experimental study.¹⁷ Furthermore, the authors used only male rats to reduce variability because the previous study found that female rat fracture model exhibits different mechanical properties.¹⁸

The inclusion criteria were healthy 3-month-old male Rattus norvegicus weighing 180–200 g. The exclusion criteria were infection, death, abnormal extremity and broken/damaged cast. The animals were acclimatized for a week in a controlled condition of 12-h light/dark cycle at the temperature of 23.6°C with gentle handling, daily cage cleaning and close monitoring; they were fed a standard chow diet with free water intake.

The rats were divided into six groups. These groups were sacrificed 14 days after fracturing the tibia by dislocating their cervical to minimize the animal suffering. The details of the groups and their interventions are as follows:

Table 1. The details of treatment in each animal model group.

Group	Intervention
Group 1	Fractured tibia, without tourniquet
Group 2	Fractured tibia, 1 hour tourniquet
Group 3	Fractured tibia, 2 hours tourniquet
Group 4	Fractured tibia, 2 hours tourniquet, 40 mg/kg BW Aloe vera gel administration
Group 5	Fractured tibia, 2 hours tourniquet, 60 mg/kg BW Aloe vera gel administration
Group 6	Fractured tibia, 2 hours tourniquet, 80 mg/kg BW Aloe vera gel administration

Tibia fracture model

The right tibia of the rat was fractured using the closed bone cutting technique.¹⁹ The animals were fasted for three hours before the procedure. The animals were anesthetized using 40 mg/kg bodyweight (BW) Ketamine HCl. The fracture was initiated by holding the rat’s leg on the proximal and distal parts using two forceps until complete fracture is achieved marked by false movement. The fracture was immobilized with long leg cast using plaster of paris (POP). The rats received analgetic medication in the form of paracetamol 10 mg/kg if there were signs of pain such as lethargy, difficulty in eating, and shivering.

Tourniquet

The tourniquet used was a 4.5 oz orthodontic rubber band with a diameter of 1/8 inch. The orthodontic rubber band was applied to one leg of the rat on the proximal thigh. The use of orthodontic rubber band in the ischemic–reperfusion injury in the murine model has been established in the previous study.²⁰ Tourniquet is often used in ischemic–reperfusion rats’ model because it provides adequate occlusion to femoral artery and its numerous collateral branches. Orthodontic rubber band is superior because it causes less soft tissue damage.²⁰

Aloe vera gel

The Aloe vera gel was created with Soxhlet method, then diluted with dimethyl sulfoxide (DMSO). The lowest dose of 40 mg/kg BW was determined using a conversion table by Laurence and Bacharach.²¹ The second dose of 60 mg/kg BW was determined as the half of the lethal dose of Aloe vera in rats (120.65 mg/kg BW/day).²² The third dose of 80 mg/kg BW was determined according to the interval between the first and second dose. The Aloe vera gel was administered three hours before application of torniquet and was given using a probe.

Superoxide dismutase (SOD) measurement

SOD is an enzyme acting as an endogenous antioxidant.⁷ In this study, the authors measured the SOD level using SOD ELISA kit. The SOD levels were measured using the serum and the tissue (bone).

Malondialdehyde (MDA) measurement

MDA is the byproduct of lipid peroxidation, a process occurring in oxidative stress.²³ The authors measured MDA level in the serum and bone tissue using Thiobarbituric acid reactive substances (TBARS) assay.

BMP-7 measurement

BMP-7 is a subfamily of Transforming Growth Factor-β (TGF-β) which has osteoinductive activity. The serum and bone tissue level of BMP-7 were measure using BMP-7 Quantikine ELISA kit.

Callus formation

The callus formed was measured manually from the harvested tibia using a standardized caliper.

Osteoblast

The number of osteoblasts in the callus tissue was measured microscopically. The number of cells was measured per high power field (HPF). The microscope used was Olympus BX-51 Dot Slide with Olympus XC10 camera.

Statistical analysis

To determine the significance of the comparison of the mean of various parameters, a hypothesis test was conducted using the One-Way ANOVA with an error rate of 5% or a confidence interval of 95%. However, before using parametric statistics, the requirements for normality and homogeneity of the data were tested first. The homogeneity of the data can be considered homogeneous because the number of samples in each group were the same. If the data was not normally distributed, the Kruskal-Wallis test was performed.

From the normality test, all data were found to be normally distributed in all groups, except for the plasma SOD variable and the number of osteoblasts. The osteoblast variable had an abnormal distribution in all groups. Variables that were normally distributed were tested using parametric statistics, namely ANOVA, while variables that were not normally distributed were tested using non-parametric statistics, namely the Kruskal-Wallis test with a p-value of <0.05 which was considered significant. Further, all pair-wise comparisons were tested using Tukey test. If the assumptions were not met, the comparison was conducted using Kruskal-Wallis test and post-hoc Dunn test. Data analysis was performed by SPSS version 25 software (RRID:SCR_002865), NY Armonk, USA. URL: http://www-01.ibm.com/software/uk/analytics/spss/.

Results

There were 18 Wistar strain rats used in this study. The measurement results of each variable in group 2 to 6 are depicted in Table 2.

Table 2. Comparison and post-hoc test of group 2-6.

Parameter	Group	N	Mean ± SD	P value
Plasma SOD (IU/mL)	2	3	44.42 ± 0.39	0.148^‡
	3	3	45.46 ± 0.64
	4	3	45.61 ± 0.17
	5	3	45.38 ± 0.76
	6	3	45.76 ± 0.51
Bone SOD (μmol/L)	2	3	13.83 ± 1.83^ab	0.036*^†
	3	3	12.98 ± 0.78^b
	4	3	14.12 ± 0.12^ab
	5	3	14.87 ± 0.11^ab
	6	3	15.83 ± 0.68^ac
Plasma MDA (ng/g)	2	3	264.67 ± 16.37^a	<0.001*^†
	3	3	239.56 ± 3.53^b
	4	3	229.33 ± 80.00^bc
	5	3	224.07 ± 0.56^bcd
	6	3	219.44 ± 1.25^bce
Bone MDA (ng/g)	2	3	377.62 ± 26.16^a	<0.001*^†
	3	3	393.78 ± 2.14^a
	4	3	270.22 ± 7.78^be
	5	3	243.66 ± 7.19^cef
	6	3	218.00 ± 1.11^df
Plasma BMP-7 (ng/g)	2	3	399.06 ± 23.26^a	0.007*^†
	3	3	393.78 ± 2.14^ade
	4	3	270.22 ± 7.78^bfg
	5	3	243.66 ± 7.19^cdfh
	6	3	218.00 ± 1.11^aegh
Bone BMP-7 (ng/g)	2	3	399.06 ± 23.26^a	<0.001*^†
	3	3	412.40 ± 19.94^a
	4	3	461.04 ± 23.39^b
	5	3	453.91 ± 5.99^ce
	6	3	439.19 ± 6.62^de
Callus (mm)	2	3	6.50 ± 0.19^a	<0.001*^†
	3	3	4.62 ± 0.25^b
	4	3	6.24 ± 0.18^a
	5	3	6.44 ± 0.38^a
	6	3	6.69 ± 0.08^a
Osteoblast (cells/HPF)	2	3	17.67 ± 0.58^ab	0.011*^‡
	3	3	6.33 ± 0.58^a
	4	3	13.33 ± 1.16^ab
	5	3	17.33 ± 0.58^ab
	6	3	19.67 ± 0.58^bc

† p-value from one-way ANOVA test.

‡ p-value from Kruskal-Wallis test.

* p-value <0.05 is considered as significant.

We observed the bone and plasma SOD level did not increase much after administration of varying dose of Aloe vera, especially in plasma SOD. Meanwhile a dose-dependent increase was observed in bone SOD measurement. Both plasma and bone MDA decreased after administration of increasing dose of Aloe vera gel.

There was no significant difference in MDA levels between the groups of rats that were given a tourniquet or not. However, the administration of Aloe vera extract was shown to reduce bone MDA levels, although there was no significant decrease in plasma MDA levels. Both plasma and bone BMP-7 were seen to increase with the administration of Aloe vera extract. However, a greater increase in bone BMP-7 was seen compared with plasma BMP-7.

After application of tourniquet, the authors observed a decrease of almost 30% of callus diameter formed (group 1 compared to group 3). Administration of Aloe vera gel increased the callus diameter close to diameter of control group. Similar reduction was observed in osteoblast cell count. In group 3, the number of osteoblasts decreased by 64.18 % compared to group 1. After administration of 40 mg/kg BW Aloe vera gel, the number of osteoblasts increased to 75% of the number in the control group. Moreover, with administration of 80 mg/kg BW Aloe vera gel, there was a 11% increase of osteoblasts number in group 6 compared to group 1.

Discussion

In fracture itself, oxidative stress can occur due to ischemic-reperfusion mechanism. In the first three days of the fracture healing phase, an ischemic phase occurs (no oxidative stress occurs). Then, at the stage of callus formation, new capillaries and proinflammatory cells will increase the production of reactive oxidative stress (ROS) that cause oxidative stress. With the increase in ROS, oxidative stress will arise which can be measured by MDA levels. Ischemic-reperfusion conditions that occur in fracture healing can be exacerbated by the use of a tourniquet. ROS cause loss of bone mass by inhibiting osteoblast differentiation and enhancing osteoclastogenesis.²⁴ The results of this study showed that there were significant differences in the variables of bone SOD, bone and plasma MDA, bone and plasma BMP-7, callus diameter and number of osteoblasts. This significant difference was also found between the groups without a tourniquet and those with a tourniquet, as well as between the groups with or without Aloe vera use. Correlation analysis showed an inverse relationship only in plasma and bone MDA variables, with significant values.

The application of a tourniquet or administration of Aloe vera did not make a significant difference to plasma or bone SOD levels. These results may indicate that the administration of antioxidants such as ethanolic extract of Aloe vera is more beneficial, if given early in the onset of ischemic-reperfusion injury, and the benefits of its administration may be reduced or even absent if the injury is long-standing. In groups 1 and 3, it can be seen that the levels of SOD, both bone and plasma, did not have a significant difference, this could mean that SOD did not play a role in ischemic-reperfusion injury conditions due to oxidative stress, although the use of tourniquets.

Both the level of plasma and bone tissue MDA increased significantly after application of tourniquet. There was also no large decrease observed with the administration of a larger dose of Aloe vera ethanolic extract. This may indicate that the effect of antioxidants such as ethanolic extract of Aloe vera is more influential in the acute phase and the decrease in bone MDA on day 14 is a physiological process. This signified that the application of tourniquet and the subsequent reperfusion during its deflation caused a marked oxidative stress in local tissue and systematically. The same result was observed in the another preliminary study by the authors.⁶

Turgut et al. used MDA levels in bone preparations to show an increase in oxidative state on bone healing in rats. MDA levels in murine model increased significantly on days 7 and 14 after tibia fracture.²⁵ Turgut et al. stated that the first three days of the fracture healing phase were similar to the ischemia phase, whereas the second and third weeks were similar to the reperfusion phase of the ischemic reperfusion mechanism.²⁵ This explains an increase in MDA levels and a decrease in SOD levels in the group after 14 days.²⁴

Bone BMP-7 showed a significant increase after administration of Aloe vera ethanolic extract at doses of 40 mg/kg BW, 60 mg/kg BW, and 80 mg/kg BW. This can indicate that the administration of Aloe vera ethanolic extract has more effect on local tissue in bone than systemic. Bone BMP-7 levels also increased with the increase in the dose of Aloe vera given. Therefore, it can be interpreted that the administration of Aloe vera ethanolic extract can induce the production of BMP-7 in bone. This is supported by previous studies discussing the physiological conditions of bone healing. Cho, et al stated that BMP-7 acts in the osteogenic phase of the bone healing process where this phase is the final phase of the bone healing process (around days 14–21), the result of this phase is the formation of a hard callus.²⁶ A previous study also has demonstrated that Aloe vera increased both number of osteoblasts and BMP-7 production.²⁷

The results of the osteoblast count were comparable to the observations made on the callus diameter. In the group of rats who were given a tourniquet for either 1 hour or 2 hours, there was a significant decrease in the number of osteoblasts when compared to group 1. ROS are known to decrease the ability of osteoblastic differentiation in experimental animal bone marrow stromal cells (BMSCs), through Wnt/β inhibition, which plays a role in stimulating osteoblast differentiation, thereby increasing osteoblast apoptosis. When lipid peroxidation occurs due to ROS, aldehyde molecules are formed, such as MDA. Increased lipid peroxidation also increases apoptosis and osteoclastogenesis. Therefore, a high MDA expression indicates an injury due to post-ischemic reperfusion that has the potential to interfere with the bone healing process.²⁴ This explains the increase in MDA as well as a decrease in the number of osteoblasts in the tourniquet-treated group. The administration of Aloe vera ethanolic extract could increase the number of osteoblasts and in group 6 (80 mg/kg BW ethanolic Aloe vera extract) the number of osteoblasts exceeded the number of controls. This was in line with previous research by Carson et al. who found that combination of whey protein and polyphenol of green tea (antioxidant) could increase osteoblast proliferation.²⁸

The use of a tourniquet causes the diameter of the callus formed after 2 weeks to be smaller. In addition, the administration of Aloe vera extract could increase the diameter of the callus until it was close to the control group. The increase in callus diameter in the group given the ethanolic extract of Aloe vera could be caused by its antioxidant property. The phenolic compound found in Aloe vera is a strong, reductant and hydrogen donor.²⁹ Other studies have demonstrated the benefit of Aloe vera in ameliorating ischemic-reperfusion injury in other organs.³⁰^,³¹ The results of our study suggest that Aloe vera gel may be used preoperatively to reduce the deleterious effect of ischemic-reperfusion injury after tourniquet application in bone fracture cases. Carson et al. also found that phenolic compound is the main antioxidant found in Aloe vera; this may explain the effect of Aloe vera in callus formation and osteoblast number in this study.³² There was a decrease in callus diameter in groups 3 when compared to group 1. These results indicate that the duration of using a tourniquet for 2 hours, the safety time to use a tourniquet, decreases the callus formation.

The strength of this study is that the results showed an improvement in callus diameter on administration of ethanolic extract of Aloe vera to the long bones (tibia) of white rats (Rattus norvegicus) in whom a tourniquet was used. This result is a very significant breakthrough in the management of ischemic-reperfusion injury. However, this study still has several limitations. Firstly, the authors did not identify the exact active polyphenolic or ethanolic compound responsible for the antioxidant activity of Aloe vera extract. The authors also did not measure the concentration of active compound in Aloe vera extract quantitatively. Moreover, there was a variation in weight of animal model used in this study. There was a possible difference in metabolic rate of Aloe vera extract and its active compound due to difference in bodyweight. This study only measured the parameters on two time points: directly after treatment and after 14 days. Future study could do measurements in more time points to better elucidate the metabolic changes imposed by ischemic-reperfusion injury and antioxidant administration. Nevertheless, this study could be used as a reference for future studies on the use of antioxidant in management of ischemic-reperfusion injury due to tourniquet use.

Conclusion

Aloe vera extract could be used as an antioxidant to reduce oxidative stress due to ischemic-reperfusion injury after application of tourniquet in fracture cases. Moreover, Aloe vera extract could improve proliferation of osteoblasts, callus formation, and BMP-7 production.

Author contributions

Thomas Erwin Christian Junus Huwae: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Hidayat Sujuti: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Retty Ratnawati: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Mohamad Hidayat: Supervision, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Data availability

Underlying data

Zenodo: Underlying data for ‘The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study’, https://doi.org/10.5281/zenodo.6258676.³³

This project contains the following underlying data:

• Raw data - Aloe vera for Ischemic-Reperfusion Injury after Tourniquet Application. (Include data for this study)

Reporting guidelines

Zenodo: ARRIVE Checklist for ‘The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study’, https://doi.org/10.5281/zenodo.6258676.³³

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

I am grateful to all my colleagues and resident in Orthopaedic and Traumatology Department, Faculty of Medicine Universitas Brawijaya - RSUD Dr. Saiful Anwar whom I have had the pleasure to work during this and other related projects.

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23. Ayala A, Muñoz MF, Argüelles S: Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxidative Med. Cell. Longev. 2014; 2014: 1–31. PubMed Abstract | Publisher Full Text
24. Prasad G, Dhillon MS, Khullar M, et al.: Evaluation of oxidative stress after fractures. A preliminary study. Acta Orthop. Belg. 2003; 69(6): 546–551. PubMed Abstract
25. Turgut A, Göktürk E, Köse N, et al.: Oxidant status increased during fracture healing in rats. Acta Orthop. Scand. 1999 [cited 2022 Feb 24]; 70(5): 487–90. PubMed Abstract | Publisher Full Text
26. Cho T-J, Gerstenfeld LC, Einhorn TA: Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2002 Mar; 17(3): 513–520. PubMed Abstract | Publisher Full Text
27. Al-Hijazi AY, AL-Mahammadawy AKAA, Altememe EI: Expression of BMP7 in Bone Tissue Treated with Aloe vera. Int. Res. J. Nat. Sci. 2015 Nov; 13(2): 39–48.
28. Carson M, Keppler JK, Brackman G, et al.: Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities. Cells Tissues Organs. 2018; 206(1–2): 106–118. PubMed Abstract | Publisher Full Text
29. Marzanna H, Dziedzic K: Hęś2019_Article_AloeVeraLWebbNaturalSourcesOfA(1).pdf. 2019; 255–65.
30. Yuksel Y, Guven M, Kaymaz B, et al.: Effects of Aloe Vera on Spinal Cord Ischemia-Reperfusion Injury of Rats. J. Investig. Surg. 2016 Dec; 29(6): 389–398. PubMed Abstract | Publisher Full Text
31. Nejatzadeh-Barandozi F: Antibacterial activities and antioxidant capacity of Aloe vera. Org. Med. Chem. Lett. 2013; 3(1): 5. PubMed Abstract | Publisher Full Text
32. López Z, Núñez-Jinez G, Avalos-Navarro G, et al.: Antioxidant and cytotoxicological effects of Aloe vera food supplements. J. Food Qual. 2017; 2017: 1–10. Publisher Full Text
33. Huwae TECJ: Data for Aloe vera for Ischemic-Reperfusion Injury after Tourniquet Application [Data set]. Zenodo. 2022. Publisher Full Text

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Version 1

VERSION 1 PUBLISHED 30 Mar 2022

Author details Author details

¹ Department of Orthopedics and Traumatology, Faculty of Medicine Universitas Brawijaya, Saiful Anwar General Hospital, Malang, East java, 65111, Indonesia
² Department of Ophthalmology, Faculty of Medicine Universitas Brawijaya, Malang, East Java, 65111, Indonesia
³ Department of Physiology, Faculty of Medicine Universitas Brawijaya, Malang, East Java, 65111, Indonesia

Thomas Erwin Christian Junus Huwae
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Mohamad Hidayat
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Hidayat Sujuti
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Retty Ratnawati
Roles: Methodology, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (1)

version 1

Published: 30 Mar 2022, 11:372

https://doi.org/10.12688/f1000research.110244.1

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Huwae TECJ, Hidayat M, Sujuti H and Ratnawati R. The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2022, 11:372 (https://doi.org/10.12688/f1000research.110244.1)

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6

Reviewer Report 17 Aug 2023

Saroj Kumar Shrestha, Jeonbuk National University, Jeonju-si, Jeollabuk-do, South Korea

Not Approved

https://doi.org/10.5256/f1000research.121826.r186509

This article is exciting to read about the ischemic reperfusion injury in fractured bone healing and the practical measure of aloe vera gel. The methods applied, and results and outcomes are excellent; however, the authors cannot express it briefly. ... Continue reading

This article is exciting to read about the ischemic reperfusion injury in fractured bone healing and the practical measure of aloe vera gel. The methods applied, and results and outcomes are excellent; however, the authors cannot express it briefly.

There are many minor grammatical mistakes; please once go over for English (grammar) checking.
I would recommend that the author add some images during the experiment like, fracture of the tibia, administration of aloe vera gel, callus diameter, and osteoblast images.

Line: 72-73: Tourniquet-related ischemia-reperfusion injury can increase superoxide synthesis and suppress endogenous antioxidant activity. This is reference number 7.
Line: 73-75: Application of tourniquet for only 2 hours showed elevated creatine phosphokinase and lactic acid in animal models, suggesting that muscle damage occurs. This should be referenced as Heppenstall et al. (1979¹).
Line 75-76: is this the authors' hypothesis, or did the authors forget to give the reference?
Is it necessary to provide the details of the time of the experiment and the name of different laboratories? I suggest that the authors remove lines 92 to 96. However, the authors can thank the various labs in the acknowledge section.

“The Ethics Committee of Faculty of Medicine, Universitas Brawijaya approved all animal protocols and all subsequent experiments with the registration number: No. 72/EC/KEPK-S3/03/2020. All animal protocols and all subsequent experiments were also carried out according to the ARRIVE guidelines and regulations”. Remove these sentences from the original position and copy them into the Animal model section.

Line 107-108: “The exclusion criteria were infection, death, abnormal extremity, and broken/damage cast.” The condition of the rats like this is before or after the experiment? If this condition of rats is before the experiment, it is better to remove this sentence from the manuscript because the authors already mentioned that he used 18 healthy three months old rats.
Remove Table 1 and write the divided groups' names and doses of Aloe vera gel into the animal model section in detail.

It is better to take a healthy control group with no fractured tibia group, so the authors can compare it to the other fractured and aloe vera gel applied groups (not necessary if not possible now).

Line 116: Shortly (main points) describe the closed bone cutting technique.
Describe detail about the aloe vera gel extraction process. How much percentage of ethanol is used for gel extraction?
Line 139: the authors used 60 mg/kg BW as the second dose. The authors mention that 60 mg/kg is half of the lethal dose of Aloe vera in rats, meaning if the authors give 60 mg/kg Aloe vera to 10 rats, 5 should have to die. But the authors do not mention any death of the rats in this experiment. Why did the authors choose these 60 mg/kg and 80mg/kg doses of aloe vera, knowing that it is toxic to rats?
Line 141: Please clearly mention the route of aloe vera gel administration in rats (not only probe).
Line 143: Describe shortly on SOD measurement. Provide the catalog number of the SOD ELISA kit. How do you prepare the bone for ELISA? Describe shortly.
Line 147: Describe the MDA level measurement from the TBARS assay very shortly.
Line 151: Describe shortly BMP-7 quantification and provide the catalog number of the BMP-7 Quantikine ELISA kit.
Line 155: Describe shortly on callus formation measurement process.
Line 158: is it possible to capture the image of osteoblast? If the authors can, please attach the pictures in the manuscript, which increases the quality of the authors' experiment and the article’s quality.
Line 181: describe the time of the experiment after the fracture of the tibia. I think this analysis was done after 14 days of a tibia fracture. The authors should have mentioned it in the result section. Further the authors can mention shortly the preparation of samples from blood and bones.
There is no description of abcdefgh in Table 2, or is it not necessary to mention it?
Line 211-216: References needed, and reference 24 is not matched for that statement.
Line 217: the authors mention no more difference between the SOD of bone and serum in line 184, but the authors mentioned a significant difference in bone SOD in line 217. Please re-check it.

Similarly, please check lines 198 and 218-219 about the level of MDA with or without a tourniquet.

Line 254-259: is these sentences fit reference 24?
Line 283: the authors measured the parameters on two-time points: directly after treatment and after 14 days. Where are these two data? I have seen only one-time point data in Table 2.

Thank you. Best wishes.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Heppenstall RB, Balderston R, Goodwin C: Pathophysiologic effects distal to a tourniquet in the dog.J Trauma. 1979; 19 (4): 234-8 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Molecular biology, Cancer, Bone Remodelilng, particulate matter, vascular smooth muscle cell, chondrogenesis, Periodontitis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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16

Reviewer Report 22 Apr 2022

Langga Sintong, Orthopaedic and Traumatology Department, Siloam Hospitals Kebon Jeruk, Jakarta, Indonesia

Approved

https://doi.org/10.5256/f1000research.121826.r129336

This is an interesting article about ischemic reperfusion injury in fractured bone healing and its prevention with Aloe vera extract. The article provides a good presentation about how Aloe vera extract can prevent ischemic reperfusion injury after tourniquet application which ... Continue reading

This is an interesting article about ischemic reperfusion injury in fractured bone healing and its prevention with Aloe vera extract. The article provides a good presentation about how Aloe vera extract can prevent ischemic reperfusion injury after tourniquet application which is commonly used in orthopaedic surgery. The specific process of how the Aloe vera can prevent those injuries is also well explained in the article. The Aloe vera administration technique is also simple and easy to implement in clinical settings. The article provides sufficient background information for readers to understand the problem. The methods and statistical analysis in this article are technically clear and sound. I hope this study can be a recommendation for future studies about preventive treatment for ischemic reperfusion injury.

For the improvement of the article, I would recommend adding the step-by-step process of how to make Aloe Vera extract. I recommend adding that information to make the research more reproducible. Then, I would recommend adding some photographs to make the article more descriptive for readers.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Orthopaedic and Traumatology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

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VERSION 1 PUBLISHED 30 Mar 2022

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Version 1 30 Mar 22	read	read

Langga Sintong, Siloam Hospitals Kebon Jeruk, Jakarta, Indonesia
Saroj Kumar Shrestha, Jeonbuk National University, Jeonju-si, South Korea

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Reviewer Report

6 Views

17 Aug 2023 | for Version 1

Saroj Kumar Shrestha, Jeonbuk National University, Jeonju-si, Jeollabuk-do, South Korea

6 Views Cite this report Responses(0)

Not Approved

This article is exciting to read about the ischemic reperfusion injury in fractured bone healing and the practical measure of aloe vera gel. The methods applied, and results and outcomes are excellent; however, the authors cannot express it briefly.

There are many minor grammatical mistakes; please once go over for English (grammar) checking.
I would recommend that the author add some images during the experiment like, fracture of the tibia, administration of aloe vera gel, callus diameter, and osteoblast images.

Line: 72-73: Tourniquet-related ischemia-reperfusion injury can increase superoxide synthesis and suppress endogenous antioxidant activity. This is reference number 7.
Line: 73-75: Application of tourniquet for only 2 hours showed elevated creatine phosphokinase and lactic acid in animal models, suggesting that muscle damage occurs. This should be referenced as Heppenstall et al. (1979¹).
Line 75-76: is this the authors' hypothesis, or did the authors forget to give the reference?
Is it necessary to provide the details of the time of the experiment and the name of different laboratories? I suggest that the authors remove lines 92 to 96. However, the authors can thank the various labs in the acknowledge section.

“The Ethics Committee of Faculty of Medicine, Universitas Brawijaya approved all animal protocols and all subsequent experiments with the registration number: No. 72/EC/KEPK-S3/03/2020. All animal protocols and all subsequent experiments were also carried out according to the ARRIVE guidelines and regulations”. Remove these sentences from the original position and copy them into the Animal model section.

Line 107-108: “The exclusion criteria were infection, death, abnormal extremity, and broken/damage cast.” The condition of the rats like this is before or after the experiment? If this condition of rats is before the experiment, it is better to remove this sentence from the manuscript because the authors already mentioned that he used 18 healthy three months old rats.
Remove Table 1 and write the divided groups' names and doses of Aloe vera gel into the animal model section in detail.

It is better to take a healthy control group with no fractured tibia group, so the authors can compare it to the other fractured and aloe vera gel applied groups (not necessary if not possible now).

Line 116: Shortly (main points) describe the closed bone cutting technique.
Describe detail about the aloe vera gel extraction process. How much percentage of ethanol is used for gel extraction?
Line 139: the authors used 60 mg/kg BW as the second dose. The authors mention that 60 mg/kg is half of the lethal dose of Aloe vera in rats, meaning if the authors give 60 mg/kg Aloe vera to 10 rats, 5 should have to die. But the authors do not mention any death of the rats in this experiment. Why did the authors choose these 60 mg/kg and 80mg/kg doses of aloe vera, knowing that it is toxic to rats?
Line 141: Please clearly mention the route of aloe vera gel administration in rats (not only probe).
Line 143: Describe shortly on SOD measurement. Provide the catalog number of the SOD ELISA kit. How do you prepare the bone for ELISA? Describe shortly.
Line 147: Describe the MDA level measurement from the TBARS assay very shortly.
Line 151: Describe shortly BMP-7 quantification and provide the catalog number of the BMP-7 Quantikine ELISA kit.
Line 155: Describe shortly on callus formation measurement process.
Line 158: is it possible to capture the image of osteoblast? If the authors can, please attach the pictures in the manuscript, which increases the quality of the authors' experiment and the article’s quality.
Line 181: describe the time of the experiment after the fracture of the tibia. I think this analysis was done after 14 days of a tibia fracture. The authors should have mentioned it in the result section. Further the authors can mention shortly the preparation of samples from blood and bones.
There is no description of abcdefgh in Table 2, or is it not necessary to mention it?
Line 211-216: References needed, and reference 24 is not matched for that statement.
Line 217: the authors mention no more difference between the SOD of bone and serum in line 184, but the authors mentioned a significant difference in bone SOD in line 217. Please re-check it.

Similarly, please check lines 198 and 218-219 about the level of MDA with or without a tourniquet.

Line 254-259: is these sentences fit reference 24?
Line 283: the authors measured the parameters on two-time points: directly after treatment and after 14 days. Where are these two data? I have seen only one-time point data in Table 2.

Thank you. Best wishes.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Heppenstall RB, Balderston R, Goodwin C: Pathophysiologic effects distal to a tourniquet in the dog.J Trauma. 1979; 19 (4): 234-8 PubMed Abstract | Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Molecular biology, Cancer, Bone Remodelilng, particulate matter, vascular smooth muscle cell, chondrogenesis, Periodontitis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

16 Views

22 Apr 2022 | for Version 1

Langga Sintong, Orthopaedic and Traumatology Department, Siloam Hospitals Kebon Jeruk, Jakarta, Indonesia

16 Views Cite this report Responses(0)

Approved

This is an interesting article about ischemic reperfusion injury in fractured bone healing and its prevention with Aloe vera extract. The article provides a good presentation about how Aloe vera extract can prevent ischemic reperfusion injury after tourniquet application which is commonly used in orthopaedic surgery. The specific process of how the Aloe vera can prevent those injuries is also well explained in the article. The Aloe vera administration technique is also simple and easy to implement in clinical settings. The article provides sufficient background information for readers to understand the problem. The methods and statistical analysis in this article are technically clear and sound. I hope this study can be a recommendation for future studies about preventive treatment for ischemic reperfusion injury.

For the improvement of the article, I would recommend adding the step-by-step process of how to make Aloe Vera extract. I recommend adding that information to make the research more reproducible. Then, I would recommend adding some photographs to make the article more descriptive for readers.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Orthopaedic and Traumatology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

[1] 1. Kumar K, Railton C, Tawfic Q: Tourniquet application during anesthesia: “What we need to know?”. J. Anaesthesiol. Clin. Pharmacol. 2016; 32(4): 424–430. PubMed Abstract | Publisher Full Text

[2] 2. Ahmed I, Chawla A, Underwood M, et al.: Tourniquet use for knee replacement surgery. Cochrane Database Syst. Rev. 2020; 2020(12). Publisher Full Text

[3] 3. Sharma JP, Salhotra R: Tourniquets in orthopedic surgery. Indian J. Orthop. 2012; 46(4): 377–383. PubMed Abstract | Publisher Full Text

[4] 4. Leurcharusmee P, Sawaddiruk P, Punjasawadwong Y, et al.: The Possible Pathophysiological Outcomes and Mechanisms of Tourniquet-Induced Ischemia-Reperfusion Injury during Total Knee Arthroplasty. Oxidative Med. Cell. Longev. 2018; 2018: 1–15. PubMed Abstract | Publisher Full Text

[5] 5. Odinsson A, Finsen V: Tourniquet use and its complications in Norway. J. Bone Joint Surg. Br. 2006 Aug; 88-B(8): 1090–1092. PubMed Abstract | Publisher Full Text

[6] 6. Huwae TECJ, Ratnawati R, Sujuti H, et al.: International Journal of Surgery Open The effect of using torniquets on fracture healing disorders: A study in wistar strain rats (Rattus norvegicus). Int. J. Surg. Open. 2020; 23: 48–52. Publisher Full Text

[7] 7. Tran TP, Tu H, Pipinos II, et al.: Tourniquet-induced acute ischemia-reperfusion injury in mouse skeletal muscles: Involvement of superoxide. Eur. J. Pharmacol. 2011 Jan; 650(1): 328–334. PubMed Abstract | Publisher Full Text

[8] 8. Pac-Soo CK, Mathew H, Ma D: Ischaemic conditioning strategies reduce ischaemia/reperfusion-induced organ injury. Br. J. Anaesth. 2015 Feb; 114(2): 204–216. PubMed Abstract | Publisher Full Text

[9] 9. Hausenloy DJ, Yellon DM: Preconditioning and postconditioning: underlying mechanisms and clinical application. Atherosclerosis. 2009 Jun; 204(2): 334–341. PubMed Abstract | Publisher Full Text

[10] 10. Huwae TECJ, Santoso ARB, Kesuma W, et al.: Reperfusion Interval as a Prevention of Lung Injury Due to Limb Ischemia–Reperfusion After Application of Tourniquet in Murine Experimental Study. Indian J. Orthop. 2020; 54(5): 704–710. PubMed Abstract | Publisher Full Text

[11] 11. Wang L, Chen B, Lin B, et al.: Methylene blue attenuates lung injury induced by hindlimb ischemia reperfusion in rats. Mediat. Inflamm. 2018; 2018: 1–8. PubMed Abstract | Publisher Full Text

[12] 12. Takhtfooladi MA, Jahanshahi A, Sotoudeh A, et al.: Effect of tramadol on lung injury induced by skeletal muscle ischemia-reperfusion: an experimental study. J. Bras. Pneumol. 2013 Jun; 39(4): 434–439. PubMed Abstract | Publisher Full Text

[13] 13. Kao MC, Jan WC, Tsai PS, et al.: Magnesium sulfate mitigates lung injury induced by bilateral lower limb ischemia-reperfusion in rats. J. Surg. Res. 2011; 171(1): e97–e106. PubMed Abstract | Publisher Full Text

[14] 14. Hsu KY, Tsai PS, Lee JJ, et al.: Platonin mitigates acute lung injury induced by bilateral lower limb ischemia-reperfusion in rats. J. Surg. Res. 2011; 167(2): e255–e262. PubMed Abstract | Publisher Full Text

[15] 15. Sehitoglu MH, Karaboga I, Kiraz A, et al.: The hepatoprotective effect of Aloe vera on ischemia-reperfusion injury in rats. North Clin. Istanbul. 2018 Oct; 6(3): 203–209. Publisher Full Text

[16] 16. Sahin H, Yener AU, Karaboga I, et al.: Protective effect of gel form of gastric gavage applicated aloe vera on ischemia reperfusion injury in renal and lung tissue. Cell. Mol. Biol. (Noisy-le-Grand). 2017 Dec; 63(12): 34–39. PubMed Abstract | Publisher Full Text

[17] 17. Prodinger PM, Bürklein D, Foehr P, et al.: Improving results in rat fracture models: Enhancing the efficacy of biomechanical testing by a modification of the experimental setup. BMC Musculoskelet. Disord. 2018; 19(1): 1–8.

[18] 18. Moreno LD, Waldman SD, Grynpas MD: Sex differences in long bone fatigue using a rat model. J. Orthop. Res. 2006 Oct; 24(10): 1926–1932.

[19] 19. Ibrahim N, Mohamad S, Mohamed N, et al.: Experimental Fracture Protocols in Assessments of Potential Agents for Osteoporotic Fracture Healing Using Rodent Models. Curr. Drug Targets. 2014; 14(14): 1642–1650. Publisher Full Text

[20] 20. Crawford RS, Hashmi FF, Jones JE, et al.: A novel model of acute murine hindlimb ischemia. Am. J. Physiol. - Hear. Circ. Physiol. 2007; 292(2): H830–H837. Publisher Full Text

[21] 21. Martina SJ: Antiplatelet Effectivity between Aspirin with Honey on Cardiovascular Disease Based on. Bleeding Time Taken on Mice. 2019. (Cvd).

[22] 22. Guo X, Mei N: Aloe vera: A review of toxicity and adverse clinical effects. J. Environ. Sci. Heal. - Part C Environ. Carcinog. Ecotoxicol. Rev. 2016; 34(2): 77–96. Publisher Full Text

[23] 23. Ayala A, Muñoz MF, Argüelles S: Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxidative Med. Cell. Longev. 2014; 2014: 1–31. PubMed Abstract | Publisher Full Text

[24] 24. Prasad G, Dhillon MS, Khullar M, et al.: Evaluation of oxidative stress after fractures. A preliminary study. Acta Orthop. Belg. 2003; 69(6): 546–551. PubMed Abstract

[25] 25. Turgut A, Göktürk E, Köse N, et al.: Oxidant status increased during fracture healing in rats. Acta Orthop. Scand. 1999 [cited 2022 Feb 24]; 70(5): 487–90. PubMed Abstract | Publisher Full Text

[26] 26. Cho T-J, Gerstenfeld LC, Einhorn TA: Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J. Bone Miner. Res. Off. J. Am. Soc. Bone Miner. Res. 2002 Mar; 17(3): 513–520. PubMed Abstract | Publisher Full Text

[27] 27. Al-Hijazi AY, AL-Mahammadawy AKAA, Altememe EI: Expression of BMP7 in Bone Tissue Treated with Aloe vera. Int. Res. J. Nat. Sci. 2015 Nov; 13(2): 39–48.

[28] 28. Carson M, Keppler JK, Brackman G, et al.: Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities. Cells Tissues Organs. 2018; 206(1–2): 106–118. PubMed Abstract | Publisher Full Text

[29] 29. Marzanna H, Dziedzic K: Hęś2019_Article_AloeVeraLWebbNaturalSourcesOfA(1).pdf. 2019; 255–65.

[30] 30. Yuksel Y, Guven M, Kaymaz B, et al.: Effects of Aloe Vera on Spinal Cord Ischemia-Reperfusion Injury of Rats. J. Investig. Surg. 2016 Dec; 29(6): 389–398. PubMed Abstract | Publisher Full Text

[31] 31. Nejatzadeh-Barandozi F: Antibacterial activities and antioxidant capacity of Aloe vera. Org. Med. Chem. Lett. 2013; 3(1): 5. PubMed Abstract | Publisher Full Text

[32] 32. López Z, Núñez-Jinez G, Avalos-Navarro G, et al.: Antioxidant and cytotoxicological effects of Aloe vera food supplements. J. Food Qual. 2017; 2017: 1–10. Publisher Full Text

[33] 33. Huwae TECJ: Data for Aloe vera for Ischemic-Reperfusion Injury after Tourniquet Application [Data set]. Zenodo. 2022. Publisher Full Text

The effect of Aloe vera ethanolic extract in preventing ischemic-reperfusion injury during long bone fracture healing after tourniquet application: An animal study

Abstract

Keywords

Introduction

Methods

Animal model

Table 1. The details of treatment in each animal model group.

Tibia fracture model

Tourniquet

Aloe vera gel

Superoxide dismutase (SOD) measurement

Malondialdehyde (MDA) measurement

BMP-7 measurement

Callus formation

Osteoblast

Statistical analysis

Results

Table 2. Comparison and post-hoc test of group 2-6.

Discussion

Conclusion

Author contributions

Data availability

Underlying data

Reporting guidelines

Acknowledgements

References

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