The effect of green mussel (<i>Perna viridis</i>) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect

Kevin Christian Tjandra; Robin Novriansyah; Edward Kurnia Setiawan Limijadi; Lydia Kuntjoro; Meita Hendrianingtyas

doi:10.12688/f1000research.132881.1

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Research Article

The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect

[version 1; peer review: 2 approved with reservations, 1 not approved]

Kevin Christian Tjandra^1,2, Robin Novriansyah^1,3, Edward Kurnia Setiawan Limijadi^1,4, Lydia Kuntjoro^1,5, Meita Hendrianingtyas^1,4

Kevin Christian Tjandra^1,2, Robin Novriansyah^1,3, [...] Edward Kurnia Setiawan Limijadi^1,4, Lydia Kuntjoro^1,5, Meita Hendrianingtyas^1,4

PUBLISHED 08 Jun 2023

Author details Author details

¹ Kariadi General Hospital, Semarang, Indonesia
² Department of Medicine, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
³ Department of Surgery, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
⁴ Department of Clinical Pathology, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
⁵ Department of Radiology, Medical Faculty, Universitas Diponegoro, Semarang, Central Java, Indonesia

Kevin Christian Tjandra
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Robin Novriansyah
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Validation, Writing – Review & Editing

Edward Kurnia Setiawan Limijadi
Roles: Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Review & Editing

Lydia Kuntjoro
Roles: Conceptualization, Data Curation, Formal Analysis, Supervision, Visualization, Writing – Review & Editing

Meita Hendrianingtyas
Roles: Conceptualization, Data Curation, Formal Analysis, Resources, Software, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: A non-union fracture is one of the most common complications arising from an untreated fracture. Bone grafts are able to fasten bone healing which can prevent and cure non-union fractures. Therefore, alternative hydroxyapatite bone grafts from waste resources are needed to increase the availability of bone grafts in the healthcare system. A bone substitute, hydroxyapatite (HA), has the ability to prevent non-union fractures. Green mussel shell contains 95.69 percent HA, allowing for an annual production of 133.97–287.07 tons per ha of HA, and is a potent alternative material in the manufacture of HA.
Methods: This research was conducted for four months using a true experimental research method with a post-test-only control group design. This study used 36 New Zealand rabbits (Oryctolagus cuniculus) which were divided into 9 groups: positive control, negative control, and intervention at weeks 2, 4 and 6 after the intervention. All groups were subjected to three general procedures: pre-surgery, surgery, and post-surgery.
Results: The findings demonstrated that green mussel shell HA has efficacy in accelerating bone healing, better than HA bovine, as compared to the 6-week negative control group and demonstrated a significant difference (p < 0.05).
Conclusions: Green mussel hydroxyapatite is proven to be able to fasten and maximize the bone healing process as fast as bovine HA, and even has higher efficacy than bovine HA.

Keywords

Hydroxyapatite, green mussel shell, bone substitute, bone healing, alkaline phosphatase

Corresponding author: Meita Hendrianingtyas

Competing interests: No competing interests were disclosed.

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

Copyright: © 2023 Tjandra KC 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: Tjandra KC, Novriansyah R, Limijadi EKS et al. The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect [version 1; peer review: 2 approved with reservations, 1 not approved]. F1000Research 2023, 12:631 (https://doi.org/10.12688/f1000research.132881.1) First published: 08 Jun 2023, 12:631 (https://doi.org/10.12688/f1000research.132881.1) Latest published: 07 Jun 2024, 12:631 (https://doi.org/10.12688/f1000research.132881.2)

Introduction

The incidence of traffic accidents has increased in Indonesia, which often cause disability in the form of fractures.¹^,² Based on data recorded by the Directorate of Traffic of the Regional Police of Central Java in 2018, the prevalence of fractures due to traffic injury is 5.5% in Indonesia.³ Data recorded by the World Health Organization (WHO) shows that there was an increase in the number of fractures from 2008 to 2009, from 13 million cases to 18 million cases along with a prevalence rate of 2.7% to 5.5%.⁴ If these conditions are not handled, the decline in quality of life and activity limitations cannot be avoided.

Fracture is a condition where there is a discontinuity of bone.⁵ Meanwhile, bones have the ability to heal.¹ Fracture healing necessitates a combination of mechanical stability from appropriate fixation, adequate bone vascularization, osteoprogenitors and growth factors from bone cells, and interaction between shattered bone fragments. Non-union fractures can occur if a combination of these conditions is not met.⁶^,⁷ As a result, bone grafts with osteogenesis, osteoinduction, and osteoconduction characteristics are required to assist in the healing of acute fractures and non-union fractures.

A substance called hydroxyapatite (HA) is frequently utilized in the development of bone substitutes. This is because HA makes up 50% of the mineral components of bone. Bone consists of 69% mineral components, 22% organic matrix, and 9% water. HA is a key component needed in the process of bone regeneration and healing.⁸ Virgin clam shells and green mussel shells are just two examples of wastes that could serve as a source of HA for bone substitutes.⁹^,¹⁰ The virgin clam shell (Anadara granosa) has been suggested as a viable material for the synthesis of HA.¹⁰ On the other hand, no recent studies related to green mussel shells have been conducted. Therefore, the potential of green mussel shells as a material for HA synthesis will be examined in this research.¹¹

According to Sangwaranatee (2016), green mussel shells contain two polymorphs of calcium carbonate (CaCO₃), namely calcite and aragonite. Most organic compounds can be found among the crystallites (intercrystalline), but some organic molecules (intercrystalline) are also intercalated in the crystal lattice. More specifically, green mussel shells consist of 95-99% CaCO₃ (calcite, aragonite, or vaterite) with lesser amounts of MgCO₃, Al (Fe₂O₃), SiO₂, Ca₃P₂O₈, CaSO₄, proteins, and mucopolysaccharides. This CaCO₃ content will then be processed into the hydroxyapatite.¹²

Indonesia is able to produce 140 – 210 tons per hectare of green mussel shell waste every year.¹³ Green mussel shells consist of 95.69% HA, so 133.97 – 287.07 tons per hectare of HA can be produced annually.¹⁴ Therefore, green mussel shells have the potential to be an alternative material in the production of HA.

Alkaline phosphatase (ALP), an enzyme that contributes to the process of bone mineralization, is produced by osteoblasts with increased activity in the third stage. As one of the elements of a complete blood count, ALP can therefore serve as a biomarker that is frequently evaluated and easily accessed to detect bone healing.¹⁵

In this research, serum ALP levels will be measured in the weeks 2, 4, and 6 to see how the distribution of green mussel shell HA affects the ALP levels. This retrieval was timed in accordance with findings from Rathwa et al. (2021), who noted that peak serum ALP levels in fracture patients occurred in the sixth week.¹⁶ This research aims to observe the effectiveness of green mussel shell HA as a bone substitute material and to provide knowledge for further research.

Methods

Ethics

Ethical clearance was issued by the Medical and Health Research Ethics Commission Faculty of Medicine, University of Diponegoro (Komite Etik Penelitian Kesehatan Fakultas Kedokteran Universitas Diponegoro), with serial number 03/EC/H/FK-UNDIP/I/2022 approved on January 11^th 2021. All methods and protocol, including the research question, key design features, and analysis plan, were performed in accordance with the relevant guidelines and regulations and the study is reported in accordance with ARRIVE guidelines. All efforts were made to ameliorate any suffering of animals. All the efforts aim for acclimatization to account for their diverse origins; each rabbit was kept separately in polycarbonate cages (0.90 0.60 0.60 m) for a week on a 12-hour light/dark cycle at a constant temperature of 25°C and humidity of 50%. Animals were routinely observed for food consumption and fecal characteristics while being fed a conventional pellet diet and drinking tap water at will.

Design

This research was carried out at the Animal Test Laboratory in the Lembah Kalipancur Tourism Village, Semarang, and the Healthy Animal Clinic Laboratory in Malang. This research was carried out for four months from September to December 2021. This study used a true experimental study with a post-test-only control group design.

Animals and materials

The experimental animals used in this study were New Zealand guinea rabbits (Oryctolagus cuniculus) obtained from rabbit breeders in Ambarawa, Semarang Regency. The inclusion criteria in this study were skeletally matured, male New Zealand rabbits (Oryctolagus cuniculus), aged 6-12 months, with a weight of 2.5 – 3 kg. The exclusion criteria in this study were there being anatomical abnormalities, signs of infection, and rabbits that died during treatment. The required sample size was calculated using a resource equation. The equation showed that each group (9 groups) should consist of 3 rabbits with 1 additional rabbit to account for drop out (10%). Thus, 36 rabbits were used in this study.

Sampling was conducted by simple random sampling to avoid bias due to variations in age and weight. Rabbits that met the inclusion and exclusion criteria were assigned randomly after being determined to be homogeneous using a simple randomization method. Then the 4 stages of blinding were conducted which included blinding during allocation, during the experiment, during the outcome assessment, and during the data analysis. After a week of adaptation, samples were obtained randomly from the group of rabbits (Oryctolagus cuniculus). Then the 36 rabbits were divided into 9 groups (4 rabbits for each group) as shown in Figure 1.

Figure 1. Study group design.

Description:

n: Sample

K₁: Negative control

K_1.1: Negative control observed sixth week after intervention

K_1.2: Negative control observed fourth week after intervention

K_1.3: Negative control observed second week after intervention

K₂: Positive control

K_2.1: Positive control observed sixth week after intervention

K_2.2: Positive control observed fourth week after intervention

K_2.3: Positive control observed second week after intervention

P: Intervention group

P₁: Intervention group observed sixth week after intervention

P₂: Intervention group observed fourth week after intervention

P₃: Intervention group observed second week after intervention

X₀: No Intervention

X₁: Bovine HA implantation in rabbit femur

X₂: Green mussel shell HA implantation in rabbit femur

Seven rabbits from seven different groups (P₁, K_1.1, P₂, K_2.2, P₃, K_2.3, K_1.3) were found dead during daily monitoring several days after the surgery with no specific cause of death. None of the humane endpoint criteria (Table 1) were noted prior to their death nor found on examination after death. Rabbits from P₁ and K_1.1 were found dead two days after the surgery, rabbits from P₂ and K_2.2 were found dead three days after the surgery, and rabbits from P₃, K_2.3, and K_1.3 were found dead five days after the surgery. The final data does not include their information. However, the data is still credible since each group achieved the minimum sample of three rabbits for each of these groups.

Table 1. Humane endpoint criteria.²⁶

Parameter	Observations
General appearance	Dehydration, decreased body weight, missing anatomy, abnormal posture,hypothermia, fractured appendage, swelling, tissue masses, prolapse, paraphimosis
Skin and fur	Discoloration, urine stain, pallor, redness, cyanosis, icterus, wound, sore,abscess, ulcer, alopecia, ruffled fur
Eyes	Exophthalmos, microphthalmia, ptosis, reddened eye, lacrimation, discharge, opacity
Nose, mouth, and head	Head tilted, nasal discharge, malocclusion, salivation
Respiration	Sneezing, dyspnea, tachypnea, rales
Urine	Discoloration, blood in urine, polyuria, anuria
Feces	Discoloration, blood in the feces, softness/diarrhea
Locomotor	Hyperactivity, coma, ataxia, circling, muscle, tremors,

Procedure

The rabbits were selected according to the inclusion criteria after each rabbit was weighed. A total of 36 rabbits that met the inclusion criteria were then adapted, given food and drink as necessary for a week. During the adaptation period, rabbits were given vitamins A, D, E (0.1 mL/kg BW IM), vitamin B complex (0.1 mL/kg BW IM), and ivermectin (0.4 mg/kg BW IM) for disease prevention. After a week-long adaptation period, the rabbits were separated each into one cage according to their group. The rabbits were monitored for their humane endpoint conditions before and after the intervention for 2 hours per day (16:00 – 18:00). Those rabbits that reached their humane endpoint will be terminated by the administration of ≥100 mg/kg of pentobarbital (lethal dose) intravascularly (IV). The humane endpoint criteria can be seen in this table below, from Montgomery (1990).

The research data obtained were primary data from the observation of blood serum alkaline phosphatase levels as a bone healing biomarker on rabbit femur bones from the treatment group compared to the negative and positive control groups. The collection of blood serum alkaline phosphatase levels as biomarkers of bone healing was carried out in week 2, week 4, and week 6 after surgery.

The green mussel shell HA used in this study is obtained from the production led by Rifky Ismail et al. at the Center of Biomechanics, Biomaterials, Biomechatronics, and Biosignal Processing (CBIOM3S).¹⁷ Then the obtained HA was powdered into a mortar and then the particle size was reduced using a stamper. In order to prevent infection, UV sterilization for ±3 hours was conducted. Before the implantation process, HA should be measured to prevent bias. The required amount of HA is calculated by the following calculation:

m = ρ \times V

m = 3.18 g / {cm}^{3} \times 3.14 \times 0.252 cm \times 0.5 cm

m = 300 mg

From the calculation, the mass of HA required for implantation of a tubular defect with a diameter of 5 mm and a depth of 5 mm is 300 mg.

There are two location options for making bone lesions in order to apply bone grafts, the metaphysis and the diaphysis. Both locations have advantages and disadvantages. There is a cushioning effect that prevents the risk of fracture due to the presence of layers of pars spongiosa and pars compacta which is an advantage of the metaphyseal section, but this section also has the disadvantage of being close to several large arteries and so there is risk of bleeding. In addition, the metaphysis has a lower volume and density than the diaphysis, making it easier to fracture.¹⁸^,¹⁹ The diaphysis location was chosen as the surgery site because it is far from the great artery and it is not easily fractured.

The surgical procedure began by shaving the hair at the incision area followed by injection of enrofloxacin 5 mg/kg IV in the rabbit’s ear through the v. auricularis lateralis as prophylactic antibiotics, and ketamine 10-40 mg/kg and acepromazine 3-5 mg/kg intramuscularly in m. longissimus dorsi caudal as the anesthetic. After that, an incision was made in the lateral area of the distal diaphysis of the femur with blade no. 22 by incising the fascia and periosteum of the rabbit femur according to the treatment group.

The procedure was conducted according to each of the experimental groups. K_1.1, K_1.2, K_1.3: make a defect in the lateral body of the femoris using a drill with a diameter of 5 mm and a depth of 5 mm. K_2.1, K_2.2, K_2.3: make a defect in lateral corpus femoris using a drill with a diameter of 5 mm and a depth of 5 mm and fill the defect with bovine HA. P₁, P₂, P₃: make a defect in the lateral part of the corpus femoris using a drill with a diameter of 5 mm and a depth of 5 mm and fill the defect with green mussel shell HA.

The fascia was sutured using absorbable catgut sutures, and the skin was sutured using non-absorbable silk. After that, the rabbits were examined until the time of data collection to know whether the rabbit showed any sign of infection, anatomical abnormalities, or died, so we could determine whether they met the exclusion criteria or not. The rabbits that showed exclusion criteria would be dropped out of the study.

The rabbits were taken care of properly by giving them food and drink as necessary until the time of blood serum data collection. The blood serum samples were taken two, four, and six weeks after surgery, depending on the intervention group of the rabbit. The intervention group is fully described in Figure 1. During this time, they were also given analgesic carbogen 2 mg/kg orally and antibiotic enrofloxacin 35mg/kg orally once per three days to keep their quality of life up.

The blood sampling was conducted by inserting needle parallel to the lateral marginal auricular vein. Once the needle reached the vein, blood will flow into the catheter. The vacutainer tube was pushed so that blood can enter the 1-2 ml tube. After collecting the required sample, the needle was immediately removed from the rabbit’s lateral marginal vein. The injected area was pressed with an alcohol swab at the end of this process.²⁰

After the blood samples were collected, the rabbits were killed by the administration of ≥100 mg/kg of pentobarbital (lethal dose) intravascularly (IV). Then, they were monitored until a lack of heartbeat was noted for >60 seconds prior to carcass disposal.

Examination of ALP is selected due to its availability in most health centers. Detection of the ALP level from blood serum was carried out using the Mindray BC2800Vet series and blood chemistry analyzer with the brand Ubio-iChem-535 at the Animal Clinic Laboratory of Healthy Animals in Malang. In a previous study, this procedure was carried out based on measuring serum ALP levels in rabbit blood through the marginal vein. Then, the alkaline phosphatase is tested using 4-nitrophenol phosphate as a substrate and 2-amino-2-methyl-1-propanol (AMP) or diethanolamine (DEA) as a buffer, with DEA yielding higher ALP results. ALP isoenzymes can be differentiated using electrophoresis, HPLC, and other techniques.²¹

Analysis

The data obtained from the biochemical assessment were described by the ratio of serum alkaline phosphatase (ALP) levels.

The Shapiro-Wilk test analyzed the normality of the data distribution. This test was chosen because the sample size in this study was <50. The data of this study were normally distributed with a p-value ≥0.05. The analysis was carried out using a one-way ANOVA test to see differences between groups because the data were normally distributed. A post hoc LSD test followed the results of the ANOVA test to find out more conclusively the relationship between variables that caused the data to be significant at the p-value <0.05. The comparison between the negative control group and the intervention group only shows a significant difference in the week 6.

In addition, data analysis was also carried out to compare the data for the week 2 and week 4; week 2 and week 6; and week 4 and week 6 using one-way ANOVA. The data of this study were considered insignificant at the p-value < 0.05.

Results

In this study, a total of 36 New Zealand rabbits (Oryctolagus cuniculus) were chosen and split into 9 groups. Each group consisted of four rabbits and were treated according to their group. The alkaline phosphatase blood serum level data was collected from the remaining 29 rabbits after 7 died before samples could be taken. The timing of data collection for rabbits was carried out according to the group, namely in the week 2, week 4, and week 6 after the intervention.

Table 2 shows the serum alkaline phosphatase (ALP) levels in rabbits in each treatment group. Differences in ALP levels were found vertically at weeks 2, 4, and 6. Horizontally, differences in ALP levels can be seen between the positive control, treatment, and negative control groups.

Table 2. Rabbit serum alkaline phosphatase level (U/L).

	Positive control (bovine)	Negative control	Treatment (green mussel shell)
Week-2^nd	44.33 (K_2.3)	72.00 (K_1.3)	60.50 (P₃)
Week-4^th	60.40 (K_2.2)	57.70 (K_1.2)	57.33 (P₂)
Week-6^th	42.17 (K_2.1)	62.53 (K_1.1)	18.65 (P₁)

Table 3 shows that the results of the one-way ANOVA test, which reported a p-value = 0.483 and a Levene value = 0.244. Because the p-value was > 0.05, this analysis test showed no significant difference in serum ALP levels at week 2, week 4, and week 6 in the bovine group (K_2.3, K_2.2, and K_2.1).

Table 3. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the positive control group (bovine) (U/L).

Bovine	Mean ± SD	p	Levene
Week-2^nd (K_2.3)	44.33 ± 19.00	0.483	0.244
Week-4^th (K_2.2)	60.40 ± 7.24
Week-6^th (K_2.1)	42.17 ± 25.91

Table 4 shows the results of the one-way ANOVA test which reported a p-value = 0.052 and a Levene value = 0.034. Because the p-value was > 0.05, this analysis test showed no significant difference in serum ALP levels at week 2, week 4, and week 6 of the green mussel shell group (P₃, P₂, and P₁).

Table 4. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the intervention group (green mussel shell) (U/L).

Green mussel	Mean ± SD	p	Levene
Week-2^nd (P₃)	60.50 ± 34.88	0.052	0.034
Week-4^th (P₂)	57.33 ± 14.72
Week-6^th (P₁)	18.65 ± 5.40

Table 5 shows the results of the one-way ANOVA test reported a p-value = 0.510 and the Levene value = 0.772. Because the p-value was > 0.05, it can be concluded that the serum ALP levels in the negative control group (K_1.3, K_1.2, and K_1.1) were not significantly different.

Table 5. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the negative control group (U/L).

Control	Mean ± SD	p	Levene
Week-2^nd (K_1.3)	72.00 ± 11.47	0.510	0.772
Week-4^th (K_1.2)	57.70 ± 17.23
Week-6^th (K_1.4)	62.53 ± 16.02

Table 6 shows that from the one-way ANOVA test results, the p-value = 0.416 and the Levene value = 0.192. Because the p-value was > 0.05, it can be concluded that the serum ALP levels in the week 2 were not significantly different.

Table 6. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-2^nd (U/L).

Week-2^nd	Mean ± SD	p	Levene
Positive control (bovine) (K_2.3)	44.33 ± 19.00	0.416	0.192
Intervention (green mussel) (P₃)	60.50 ± 34.88
Negative control (K_1.3)	72.00 ± 11.47

Table 7 shows that the results of the one-way ANOVA test reported a p-value = 0.959 and the Levene value = 0.454. Because the p-value was > 0.05, it can be concluded that the serum ALP levels in the week 4 were not significantly different.

Table 7. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-4^th (U/L).

Week-4^th	Mean ± SD	p	Levene
Positive control (bovine) (K_2.2)	60.40 ± 7.24	0.959	0.454
Intervention (green mussel) (P₂)	57.33 ± 14.72
Negative control (K_1.2)	57.70 ± 17.23

Table 8 shows that from the one-way ANOVA test results, the p-value = 0.030 and the Levene value = 0.087. Because the p-value was < 0.05 and a Levene value > 0.05, it can be concluded that the serum levels of ALP in the positive control, treatment, and negative control groups at week 6 had significant differences and were homogeneous. The test was continued with the LSD post hoc test to find out the differences between the treatment groups.

Table 8. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6^th (U/L).

Week 6^th	Mean ± SD	p	Levene
Positive control (bovine) (K_2.1)	42.17 ± 25.91	0.030	0.087
Intervention (green mussel) (P₁)	18.65 ± 5.40
Negative control (K_1.1)	62.53 ± 16.02

Table 9 above shows that the results of the post hoc LSD test reported the positive control group (bovine) (K_2.1) against the treatment group (green mussel shells) (P₁) and the negative control group (K_1.1) were not significantly different, while the treatment group (green mussel shells) (P₁) against the negative control group (K_1.1) had significant results.

Table 9. The results of the LSD post hoc test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6^th (U/L).

Group		p	Description
I	II	p	Description
Positive control (bovine) (K_2.1)	Intervention (green mussel) (P₁)	0.107	Not significant
	Negative control (K_1.1)	0.178	Not significant
Intervention (green mussel) (P₁)	Negative control (K_1.1)	0.011	Significant

Discussion

The osteoconductive matrix is one of the elements that influences the bone healing process. It serves as a scaffold or mineralizing agent for bone and helps in the adherence of osteoconductive and osteogenic cells to the fracture site. Hydroxyapatite is one form of an osteoconductive matrix (HA). Hydroxyapatite is a substance made up of calcium (Ca²⁺), hydroxy (OH-), and phosphate (PO₄^3-) ions that form a crystal structure with the chemical formula Ca₅(PO4)₃(OH). The body will naturally generate hydroxyapatite from Ca²⁺ ions in the blood and inorganic phosphate compounds (P_i or PO₄^3-). Compound P_i is generated by dephosphorylating organic phosphate compounds (PP_i), which are phosphates that happen at the ALP dephosphorylation process. As a result, the presence of a hydroxyapatite scaffold reduces ALP activity since the body no longer needs to synthesize hydroxyapatite from PP_i.¹⁶^,²²

Because of that, ALP levels in a bone healing union will be normal or slightly elevated since the hydroxyapatite in green mussel shells functions well as a scaffold. In contrast to non-union fractures, the bone healing process takes longer, and more phosphate is required to compensate for the failure of bone healing, causing ALP levels to grow higher than in union fractures. This condition serves as the study’s measurement basis.¹⁶^,²² However, ALP does not only play a role in the bone healing process, but also plays a role in other organ systems, such as hepatobiliary, genitourinary, and gastrointestinal.²²^,²³ As a result, it is important to ensure that these body functions are in good condition so that false negatives or false positives do not occur.

This study aims to determine the effect of green mussel shell hydroxyapatite on blood alkaline phosphatase (ALP) levels in bone grafting procedures in New Zealand rabbits. The total sample was 36 New Zealand rabbits which were then divided into 9 groups. Following this, observations were made in the groups of negative control, positive control groups, and intervention groups in the weeks 2, 4, and 6. Using a surgical procedure and drilling with a width and depth of 5 mm, all groups produced lesions on the femoral diaphysis. The treatment group received green mussel shell hydroxyapatite (HA), while the negative control group did not get any HA as a follow-up intervention. The positive control group received bovine hydroxyapatite (HA). Drill lesions were made to develop the femoral diaphysis to replicate a fracture and to provide a suitable area to apply HA to the cavity formed by the drilling process. In this experiment, an effective dose of bone healing was up to 300 mg of the bone graft.¹⁰

This experiment had a number of methodological limitations, including fractures in the rabbits and post-interventional infections that were not equally exposed to all of the rabbits. This may impact the trial outcomes by increasing the time it takes for bones to recover, which could prevent all treatment groups from showing significant results between the weeks 2, 4, and 6. Even though all groups received an appropriate dose of 300 mg at the area of the lesion, the prolonged bone healing time meant that the test between the positive control group and the negative control group did not produce significant results in the week 6.

According to data analysis, the only test that produced significant results with a p-value less than 0.05 on the post-hoc LSD test was the comparison between the week 6 negative control group (K_1.1) and the week 6 treatment group (P₁). This demonstrates the effectiveness of green mussel shell HA in accelerating and maximizing the healing of bone fractures. however, it could not detect a significant difference between the week 2 and 4. This shows that the week 6, when ALP activity is at its peak, is the optimal week to observe how HA treatment affects the bone-healing process in femoral fractures as compared to weeks 2 and 4.¹⁶

Additionally, even though there were no statistically significant differences between the week 6 positive control group (K_2.1) and the week 6 negative control group (K_1.1) in the test, K_2.1’s ALP level was lower than K₁’s. This demonstrates that bovine HA can speed up bone repair, but its effectiveness is lower than that of HA from green mussel shells.

A comparison test of the serum ALP levels at the weeks 2, 4, and 6 in each treatment group’s results did not reveal any significant differences. This indicates that while ALP activity varied at weeks 2, 4, and 6, the differences were not statistically significant. This means that the effect of giving HA works evenly in the weeks 2, 4, and 6.

Conclusion

Green mussel hydroxyapatite (HA) is proven able to fasten and maximize the bone healing process as fast as bovine HA and even has higher efficacy than bovine HA.

Data availability

Underlying data

Mendeley Data: Green Mussel Shell Hydroxyapatite ALP Data Set, https://data.mendeley.com/datasets/gvhpb34kkr/1.²⁴

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

Reporting guidelines

ARRIVE checklist for ‘The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect’, https://doi.org/10.17632/vyw756k44g.1.

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

References

1. Andrzejowski P, Giannoudis PV: The ‘diamond concept’ for long bone non-union management. J. Orthop. Traumatol. 2019; 20(1): 1–3.
2. Qamar N, Aswari A: Healing or Hurting: development of highway public transportation technology. J. Dinamika Huk. 2018; 18(3): 319–328.
3. Kementerian Kesehatan RI, Hasil Riset Kesehatan Dasar (Riskesdas): 2018. Badan Penelitian dan Pengembangan Kesehatan Kementerian RI.2018.
4. Amin S, Achenbach SJ, Atkinson EJ, et al.: Trends in fracture incidence: a population-based study over 20 years. J. Bone Miner. Res. 2014; 29(3): 581–589. PubMed Abstract | Publisher Full Text | Free Full Text
5. Noorisa R, Apriliwati D, Aziz A, et al.: The characteristic of patients with femoral fracture in Department Of Orthopaedic And Traumatology RSUD Dr. Soetomo Surabaya 2013–2016. J. Orthop. Traumatol. Surabaya. 2017; 6(1): 1–1.
6. Babcock S, Kellam JF: Hip fracture nonunions: diagnosis, treatment, and special considerations in elderly patients. Adv. Orthop. 2018; 2018: 1–11. PubMed Abstract | Publisher Full Text | Free Full Text
7. Frölke JP, Patka P: Definition and classification of fracture non-unions. Injury. 2007; 38: S19–S22. PubMed Abstract | Publisher Full Text
8. Kattimani VS, Kondaka S, Lingamaneni KP: Hydroxyapatite–-Past, present, and future in bone regeneration. Bone Tissue Regen. Insights. 2016; 7: BTRI.S36138–BTRI.S36119. Publisher Full Text
9. Bhatt RA, Rozental TD: Bone graft substitutes. Hand Clin. 2012; 28(4): 457–468. Publisher Full Text
10. Vecchio KS, Zhang X, Massie JB, et al.: Conversion of bulk seashells to biocompatible hydroxyapatite for bone implants. Acta Biomater. 2007; 3(6): 910–918. PubMed Abstract | Publisher Full Text
11. Dhanaraj K, Suresh G: Conversion of waste sea shell (Anadara granosa) into valuable nanohydroxyapatite (nHAp) for biomedical applications. Vacuum. 2018; 152: 222–230. Publisher Full Text
12. Sangwaranatee NW, Teanchai K, Kongsriprapan S, et al.: Characterization and analyzation of chitosan powder from Perna Viridis shell. Materials Today: Proceedings. 2018; 5(6): 13922–13925.
13. Fitriah E, Maryuningsih Y, Roviati E: Pemanfaatan daging dan cangkang kerang hijau (Perna viridis) sebagai bahan olahan pangan tinggi kalsium. Proceeding of The URECOL. 2018; pp. 412–423.
14. Liemawan AE, Tavio T, Raka IG: Pemanfaatan limbah kerang hijau (Perna Viridis L.) sebagai bahan campuran kadar optimum agregat halus pada beton mix design dengan metode substitusi. Jurnal Teknik ITS. 2015; 4(1): F128–F133.
15. Nazht HH, Omara RA, Al Dahhan MR, et al.: Estimation of alkaline phosphatase enzymes level in the femoral transverse fractures healing in rabbits.2019. Publisher Full Text
16. Rathwa HS, Verma T, Chavali VH: Assessment of union in fractures: Role of Serum Alkaline Phosphatase and Ultrasonography. J. Clin. Orthop. Trauma. 2021; 14: 94–100. PubMed Abstract | Publisher Full Text | Free Full Text
17. Ismail R, Laroybafih MB, Fitriyana DF, et al.: The effect of hydrothermal holding time on the characterization of hydroxyapatite synthesized from green mussel shells. Journal of Advanced Research in Fluid Mechanics and Thermal Sciences. 2021; 80(1): 84–93. Publisher Full Text
18. Ajayi IE, Shawulu JC, Zachariya TS, et al.: Osteomorphometry of the bones of the thigh, crus and foot in the New Zealand white rabbit (Oryctolagus cuniculus). Ital. J. Anat. Embryol. 2012; 117(3): 125–134. PubMed Abstract
19. Bagi CM, Berryman E, Moalli MR: Comparative bone anatomy of commonly used laboratory animals: implications for drug discovery. Comp. Med. 2011; 61(1): 76–85. PubMed Abstract
20. Nazht HH: Estimation the alkaline phosphatase (ALP) level in partial hepatectomy rabbits. Al-Qadisiyah Journal of Veterinary Medicine Sciences. 2015; 14(1): 10–14.
21. Washington IM, Van Hoosier G: Clinical Biochemistry and Hematology. The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents. 2012; pp. 57–116. Publisher Full Text
22. Orimo H: The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J. Nippon Med. Sch. 2010; 77(1): 4–12. PubMed Abstract | Publisher Full Text
23. Stigbrand T, Wahren B: Alkaline Phosphatases. Serological Cancer. Markers. 1992; 11: 135. Publisher Full Text
24. Tjandra KC, Novriansyah R, Limijadi EKS, et al.: The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect. Mendeley Data. 2023; V1. Publisher Full Text
25. Tjandra KC, Novriansyah R, Limijadi EKS, et al.: The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect. Mendeley Data. 2023; V1. Publisher Full Text

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Author details Author details

¹ Kariadi General Hospital, Semarang, Indonesia
² Department of Medicine, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
³ Department of Surgery, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
⁴ Department of Clinical Pathology, Faculty of Medicine, Universitas Diponegoro, Semarang, Central Java, Indonesia
⁵ Department of Radiology, Medical Faculty, Universitas Diponegoro, Semarang, Central Java, Indonesia

Kevin Christian Tjandra
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Robin Novriansyah
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Validation, Writing – Review & Editing

Edward Kurnia Setiawan Limijadi
Roles: Methodology, Project Administration, Resources, Software, Validation, Visualization, Writing – Review & Editing

Lydia Kuntjoro
Roles: Conceptualization, Data Curation, Formal Analysis, Supervision, Visualization, Writing – Review & Editing

Meita Hendrianingtyas
Roles: Conceptualization, Data Curation, Formal Analysis, Resources, Software, Supervision, Validation, 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.

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https://doi.org/10.12688/f1000research.132881.2

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https://doi.org/10.12688/f1000research.132881.1

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Tjandra KC, Novriansyah R, Limijadi EKS et al. The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect [version 1; peer review: 2 approved with reservations, 1 not approved]. F1000Research 2023, 12:631 (https://doi.org/10.12688/f1000research.132881.1)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

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Reviewer Report 06 Sep 2023

Maria Apriliani Gani, Pharmacology-Clinical Pharmacy, Bandung Institute of Technology, Bandung, West Java, Indonesia; Universitas Airlangga, Surabaya, East Java, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.145833.r200155

The manuscript “The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect” was aimed to observe the effectiveness of green mussel shell HA as a bone substitute material.

... Continue reading

The manuscript “The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect” was aimed to observe the effectiveness of green mussel shell HA as a bone substitute material.

Before being indexed, the article must go through a major revision, including data addition and extensive English editing.

Abstract:

It is recommended to summarize the background section in the abstract and detail the aim of the study.
In the method section of the abstract, kindly briefly mention the kind of evaluation that was used in the study (histology, etc.).
The results section in the abstract should be written in the past tense.

Introduction:

Kindly check and revise the grammar in this section. Please be aware of when to use past tense etc.
“If these conditions are not handled, the decline in quality of life and activity limitations cannot be avoided.” Kindly add the data based on another study regarding quality of life and fracture incidence rate before this sentence.
“As a result, bone grafts with osteogenesis, osteoinduction, and osteoconduction characteristics are required to assist in the healing of acute fractures and non-union fractures.” Please add the reference for this.
“A substance called hydroxyapatite (HA)”. I recommend the authors to change the word “substance” to “material.”
“Virgin clam shells and green mussel shells are just two examples of wastes that could serve as a source of HA for bone substitutes.” Kindly delete the word “just.”
The author should explain why virgin clam shells and green mussel shells are recommended to be used as HA sources. What are the differences between them with other natural sources of HA?
The author should detail the hypothesis of the use of green mussel shells in the bone healing process, in terms of the chemical and physical characteristics of this material.
“Alkaline phosphatase (ALP), an enzyme that contributes to the process of bone mineralization, is produced by osteoblasts with increased activity in the third stage.” Delete the “third stage” or keep it with prior explaining about bone healing stages.
“As one of the elements of a complete blood count, ALP can therefore serve as a biomarker that is frequently evaluated and easily accessed to detect bone healing.” The authors should detail the correlation of bone healing processes with ALP serum levels not only in humans but also in animal models. Does it really have a positive correlation of not statistically differ based on other papers?

Methods:

I recommend the authors summarize the methods section.
“The inclusion criteria in this study were skeletally matured, male New Zealand rabbits (Oryctolagus cuniculus), aged 6-12 months, with a weight of 2.5 – 3 kg. The exclusion criteria in this study were there being anatomical abnormalities, signs of infection, and rabbits that died during treatment.” The inclusion and exclusion criteria mean that any criteria chosen before the study begins. Rabbits who died during the treatments are not included in the criteria. Any animals that died during the treatment must be noted, observed, and reported in the manuscript.
There was a high number of deaths of the animal and it is a must that the authors should clearly explain the cause of death in the manuscript.
Does the defect model present the “non-union fractures”? The authors should mention the criteria of non-union fracture and match it with the defect model that was created in the study.
The authors treated the rabbit with HAs in powder form. How do the authors ensure that the material can serve as a scaffold in the defective tissue? Since HAs may combine with the blood in the defect site it makes the “scaffold” and the treatment fail.
Please explain why the authors use bovine HA as the positive control, as well as the company of the bovine HA that was used in this study.
Table 1 is not needed to be included in the manuscript, it is enough to mention and cite the source.
The authors should detail the product code and company that provided the ALP kit.

Results:

Table 2-9 came from the same data set, which was the ALP levels. These tables can be present in one graph only.
The authors must add another data set in the manuscript, including:

Characteristics of green mussel shells HA and bovine HA.
Cytotoxic test for the HAs.
Other in vivo evaluation results such as radiology and histology to support the current findings.

Discussion:

The authors should detail the concept of ALP levels in each timeline in the bone healing process. When usually the ALP reaches its peaks etc. and why.

“As a result, the presence of a hydroxyapatite scaffold reduces ALP activity since the body no longer needs to synthesize hydroxyapatite from PPi.” This process occurs in the bone tissue. The authors should include the correlation of the tissue and serum ALP levels.
Since the HA came from shells, is there any potential allergic reactivity of the developed material in the human body?

References:

Please update the references with the up-to-date articles.

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?

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: Biomaterials, biomedical pharmacy, pharmacology.

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, however I have significant reservations, as outlined above.

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Reviewer Report 06 Sep 2023

Happy Kurnia Permatasari, Department of Biochemistry and Biomolecular, Faculty of Medicine, Brawijaya University, Malang, Indonesia

Fahrul Nurkolis, Biological Sciences, State Islamic University of Sunan Kalijaga (UIN Sunan Kalijaga Yogyakarta), Yogyakarta, Yogyakarta, 55281, Indonesia

Rudy Kurniawan, Graduate School of Medicine, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia

Approved with Reservations

https://doi.org/10.5256/f1000research.145833.r200163

We have fully and carefully evaluated this manuscript, "The effect of green mussel (Perna viridis) shells' hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect" by Tjandra et al.

Here are our comments:

... Continue reading

We have fully and carefully evaluated this manuscript, "The effect of green mussel (Perna viridis) shells' hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect" by Tjandra et al.

Here are our comments:

1. First of all, we agree with the other reviewer who requires the authors to re-edit the abstract section. The aims of this research are not yet visible in the abstract. This "better than HA bovine" refers to control? Or which intervention? Because it is not mentioned in the abstract methods, but appears in the results.

2. Introduction:

Provide the latest data and a wider area, it is stated that the authors adapted data from "Regional Police of Central Java in 2018". This is a small part of Indonesia and does not reflect the urgency in the world; and some data got "2008 to 2009", please fix it to be more updated.
The aim of the study must also be addressed from the perspective of describing the contribution to the field under analysis and the elements of scientific novelty presented, as the last, separate paragraph of this section, to make it more easily visible. Develop it better. What differentiates your paper from others on the same topic? Give a reason for interest in this paper.

3. Methods:

Register this research protocol at https://preclinicaltrials.eu and mention the registration number in the methods section; it can be registered at any time and for free, this is to increase the readability value of this article.
Writing the Latin name Oryctolagus cuniculus at the beginning can be long, but from now on it must be O. cuniculus; and just write the Latin name without "Rabbits"; to be more efficient.
Analysis: Data analysis is processed using what software? With what CI? It should be clear and detailed.
How is the sample size calculated? What formula do the authors use?
What is the HA dosage given based on?

4. Results:
It would be more valid to display tissue histology figure observation data from Animal Experimental to prove the authors' claim to the conclusion that "Green mussel hydroxyapatite (HA) is proven able to fasten and maximize the bone healing process as fast as bovine HA and even has higher efficacy than bovine HA."

5. Discussion:
A possible mechanism for the bone healing process must be discussed in a comprehensive manner, due to the long research, this raises the question of whether Green mussel hydroxyapatite (HA) really contributes to the healing process or if it is due to the healing process over time.

6. Very brief conclusion, elaborate it with the findings that have been observed in the research.

We conclude that this article needs to be extensively revised.

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?

No
If applicable, is the statistical analysis and its interpretation appropriate?

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

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: In silico, in-vitro and in-vivo studies; natural biomaterials; nutraceuticals; functional food; biochemistry and biomolecular; natural product

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

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Reviewer Report 24 Aug 2023

George J Dias, University of Otago, Dunedin, New Zealand

Not Approved

https://doi.org/10.5256/f1000research.145833.r195140

The Abstract needs to be improved.

Page 5: Seven rabbits from seven different groups (P1, K1.1, P2, K2.2, P3, K2.3, K1.3) were found dead during daily monitoring several days after the surgery with no specific cause of death.
... Continue reading

The Abstract needs to be improved.

Page 5: Seven rabbits from seven different groups (P1, K1.1, P2, K2.2, P3, K2.3, K1.3) were found dead during daily monitoring several days after the surgery with no specific cause of death.
This is a relatively high number of deaths, 7 out of 36 - ~20%. Very surprising that cause of death such as infection, haemorrhage was not determined?

..., UV sterilization for 3 hours was conducted.
I consider this sterilization method to be inadequate for a material to be surgically implanted. Because UV light will only disinfect the surface and will not have an effect of the interior of the implant particles. Authors need to provide evidence about how this sterilization method was selected using general surgical principals.

Page 6: ... using a drill.
Give more details of the bone drilling process. What was the speed of the motor? The type and size of the bur? Was the bur irrigated during drilling?

fill the defect with green mussel shell HA.
What was the form of HA? Was it a powder? If so what is the size of the particles?

The blood sampling was conducted by inserting needle parallel to the lateral marginal auricular vein. Once the needle reached the vein, blood will flow into the catheter. The vacutainer tube was pushed so that blood can enter the 1-2 ml tube. After collecting the required sample, the needle was immediately removed from the rabbit’s lateral marginal vein. The injected area was pressed with an alcohol swab at the end of this process.
It is not necessary to provide minute details on venopuncture. It would have been preferable if this much of details were provided for the surgical technique of HA implantation.

Examination of ALP is selected due to its availability in most health centers.
Question is blood samples tested in Animal Health Centres are that of common companion animals such as dogs and cats. Therefore, can testing ALP in rabbit blood in a health centre be accurate?

Page 9: As a result, the presence of a hydroxyapatite scaffold reduces ALP activity since the body no longer needs to synthesize hydroxyapatite from PPi.
It can also be argued that the low ALP level indicate that the rate of bone remodelling is slow, leading to the rate of new bone formation to be also being low.

This experiment had a number of methodological limitations, including fractures in the rabbits and post-interventional infections that were not equally exposed to all of the rabbits. This may impact the trial outcomes by increasing the time it takes for bones to recover, which could prevent all treatment groups from showing significant results between the weeks 2, 4, and 6. Even though all groups received an appropriate dose of 300 mg at the area of the lesion, the prolonged bone
healing time meant that the test between the positive control group and the negative control group did not produce significant results in the week 6.
If most of the animals in this study experienced delayed healing of surgical sites due to infections, then the outcome of the complete investigation is brought into question?

..... negative control group (K1.1)
In the negative control, although there was no implant material in the surgical site, the bone defect would not have been empty, it would have been filled with blood clot that eventually got organised, leading to bone healing with new bone formation. Therefore, the significant difference between negative group and the treatment group may indicate that the green mussel shell hydroxyapatite may not be effective as the natural blood clot?

A comparison test of the serum ALP levels at the weeks 2, 4, and 6 in each treatment group’s results did not reveal any significant differences. This indicates that while ALP activity varied at weeks 2, 4, and 6, the differences were not statistically significant. This means that the effect of giving HA works evenly in the weeks 2, 4, and 6.
It is very curious why the authors did not take bone tissue biopsies from the surgical sites at these time points and investigate the histology, and compare these findings with that of the ALP values?

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?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biomaterials, in-vitro and in-vovo investigations.Implants for surgical applications.

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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George J Dias, University of Otago, Dunedin, New Zealand
Happy Kurnia Permatasari, Brawijaya University, Malang, Indonesia

Fahrul Nurkolis, State Islamic University of Sunan Kalijaga (UIN Sunan Kalijaga Yogyakarta), Yogyakarta, Indonesia

Rudy Kurniawan, Hasanuddin University (Ringgold ID: 64739), Makassar, Indonesia
Maria Apriliani Gani, Bandung Institute of Technology, Bandung, Indonesia; Universitas Airlangga, Surabaya, Indonesia

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19 Jun 2024 | for Version 2

Happy Kurnia Permatasari, Department of Biochemistry and Biomolecular, Faculty of Medicine, Brawijaya University, Malang, Indonesia

Fahrul Nurkolis, Biological Sciences, State Islamic University of Sunan Kalijaga (UIN Sunan Kalijaga Yogyakarta), Yogyakarta, Yogyakarta, 55281, Indonesia

Rudy Kurniawan, Graduate School of Medicine, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia

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Approved

The authors have made significant changes to the manuscript, which has positively affected its quality.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

In silico, in-vitro and in-vivo studies; natural biomaterials; nutraceuticals; functional food; biochemistry and biomolecular; natural product

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

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8 Views

18 Jun 2024 | for Version 2

George J Dias, University of Otago, Dunedin, New Zealand

8 Views Cite this report Responses(0)

Approved

I am happy with the revisions made by the authors. I consider that this manuscript is ready to be Indexed.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomaterials, in-vitro and in-vovo investigations.Implants for surgical applications.

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.

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06 Sep 2023 | for Version 1

Maria Apriliani Gani, Pharmacology-Clinical Pharmacy, Bandung Institute of Technology, Bandung, West Java, Indonesia; Universitas Airlangga, Surabaya, East Java, Indonesia

8 Views Cite this report Responses(0)

Approved With Reservations

The manuscript “The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect” was aimed to observe the effectiveness of green mussel shell HA as a bone substitute material.

Before being indexed, the article must go through a major revision, including data addition and extensive English editing.

Abstract:

It is recommended to summarize the background section in the abstract and detail the aim of the study.
In the method section of the abstract, kindly briefly mention the kind of evaluation that was used in the study (histology, etc.).
The results section in the abstract should be written in the past tense.

Introduction:

Kindly check and revise the grammar in this section. Please be aware of when to use past tense etc.
“If these conditions are not handled, the decline in quality of life and activity limitations cannot be avoided.” Kindly add the data based on another study regarding quality of life and fracture incidence rate before this sentence.
“As a result, bone grafts with osteogenesis, osteoinduction, and osteoconduction characteristics are required to assist in the healing of acute fractures and non-union fractures.” Please add the reference for this.
“A substance called hydroxyapatite (HA)”. I recommend the authors to change the word “substance” to “material.”
“Virgin clam shells and green mussel shells are just two examples of wastes that could serve as a source of HA for bone substitutes.” Kindly delete the word “just.”
The author should explain why virgin clam shells and green mussel shells are recommended to be used as HA sources. What are the differences between them with other natural sources of HA?
The author should detail the hypothesis of the use of green mussel shells in the bone healing process, in terms of the chemical and physical characteristics of this material.
“Alkaline phosphatase (ALP), an enzyme that contributes to the process of bone mineralization, is produced by osteoblasts with increased activity in the third stage.” Delete the “third stage” or keep it with prior explaining about bone healing stages.
“As one of the elements of a complete blood count, ALP can therefore serve as a biomarker that is frequently evaluated and easily accessed to detect bone healing.” The authors should detail the correlation of bone healing processes with ALP serum levels not only in humans but also in animal models. Does it really have a positive correlation of not statistically differ based on other papers?

Methods:

I recommend the authors summarize the methods section.
“The inclusion criteria in this study were skeletally matured, male New Zealand rabbits (Oryctolagus cuniculus), aged 6-12 months, with a weight of 2.5 – 3 kg. The exclusion criteria in this study were there being anatomical abnormalities, signs of infection, and rabbits that died during treatment.” The inclusion and exclusion criteria mean that any criteria chosen before the study begins. Rabbits who died during the treatments are not included in the criteria. Any animals that died during the treatment must be noted, observed, and reported in the manuscript.
There was a high number of deaths of the animal and it is a must that the authors should clearly explain the cause of death in the manuscript.
Does the defect model present the “non-union fractures”? The authors should mention the criteria of non-union fracture and match it with the defect model that was created in the study.
The authors treated the rabbit with HAs in powder form. How do the authors ensure that the material can serve as a scaffold in the defective tissue? Since HAs may combine with the blood in the defect site it makes the “scaffold” and the treatment fail.
Please explain why the authors use bovine HA as the positive control, as well as the company of the bovine HA that was used in this study.
Table 1 is not needed to be included in the manuscript, it is enough to mention and cite the source.
The authors should detail the product code and company that provided the ALP kit.

Results:

Table 2-9 came from the same data set, which was the ALP levels. These tables can be present in one graph only.
The authors must add another data set in the manuscript, including:

Characteristics of green mussel shells HA and bovine HA.
Cytotoxic test for the HAs.
Other in vivo evaluation results such as radiology and histology to support the current findings.

Discussion:

The authors should detail the concept of ALP levels in each timeline in the bone healing process. When usually the ALP reaches its peaks etc. and why.

“As a result, the presence of a hydroxyapatite scaffold reduces ALP activity since the body no longer needs to synthesize hydroxyapatite from PPi.” This process occurs in the bone tissue. The authors should include the correlation of the tissue and serum ALP levels.
Since the HA came from shells, is there any potential allergic reactivity of the developed material in the human body?

References:

Please update the references with the up-to-date articles.

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?

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

Biomaterials, biomedical pharmacy, pharmacology.

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, however I have significant reservations, as outlined above.

Respond to this report

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Reviewer Report

9 Views

06 Sep 2023 | for Version 1

Happy Kurnia Permatasari, Department of Biochemistry and Biomolecular, Faculty of Medicine, Brawijaya University, Malang, Indonesia

Fahrul Nurkolis, Biological Sciences, State Islamic University of Sunan Kalijaga (UIN Sunan Kalijaga Yogyakarta), Yogyakarta, Yogyakarta, 55281, Indonesia

Rudy Kurniawan, Graduate School of Medicine, Hasanuddin University (Ringgold ID: 64739), Makassar, South Sulawesi, Indonesia

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Approved With Reservations

We have fully and carefully evaluated this manuscript, "The effect of green mussel (Perna viridis) shells' hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect" by Tjandra et al.

Here are our comments:

1. First of all, we agree with the other reviewer who requires the authors to re-edit the abstract section. The aims of this research are not yet visible in the abstract. This "better than HA bovine" refers to control? Or which intervention? Because it is not mentioned in the abstract methods, but appears in the results.

2. Introduction:

Provide the latest data and a wider area, it is stated that the authors adapted data from "Regional Police of Central Java in 2018". This is a small part of Indonesia and does not reflect the urgency in the world; and some data got "2008 to 2009", please fix it to be more updated.
The aim of the study must also be addressed from the perspective of describing the contribution to the field under analysis and the elements of scientific novelty presented, as the last, separate paragraph of this section, to make it more easily visible. Develop it better. What differentiates your paper from others on the same topic? Give a reason for interest in this paper.

3. Methods:

Register this research protocol at https://preclinicaltrials.eu and mention the registration number in the methods section; it can be registered at any time and for free, this is to increase the readability value of this article.
Writing the Latin name Oryctolagus cuniculus at the beginning can be long, but from now on it must be O. cuniculus; and just write the Latin name without "Rabbits"; to be more efficient.
Analysis: Data analysis is processed using what software? With what CI? It should be clear and detailed.
How is the sample size calculated? What formula do the authors use?
What is the HA dosage given based on?

4. Results:
It would be more valid to display tissue histology figure observation data from Animal Experimental to prove the authors' claim to the conclusion that "Green mussel hydroxyapatite (HA) is proven able to fasten and maximize the bone healing process as fast as bovine HA and even has higher efficacy than bovine HA."

5. Discussion:
A possible mechanism for the bone healing process must be discussed in a comprehensive manner, due to the long research, this raises the question of whether Green mussel hydroxyapatite (HA) really contributes to the healing process or if it is due to the healing process over time.

6. Very brief conclusion, elaborate it with the findings that have been observed in the research.

We conclude that this article needs to be extensively revised.

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?

No
If applicable, is the statistical analysis and its interpretation appropriate?

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

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

In silico, in-vitro and in-vivo studies; natural biomaterials; nutraceuticals; functional food; biochemistry and biomolecular; natural product

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

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Reviewer Report

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24 Aug 2023 | for Version 1

George J Dias, University of Otago, Dunedin, New Zealand

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Not Approved

The Abstract needs to be improved.

Page 5: Seven rabbits from seven different groups (P1, K1.1, P2, K2.2, P3, K2.3, K1.3) were found dead during daily monitoring several days after the surgery with no specific cause of death.
This is a relatively high number of deaths, 7 out of 36 - ~20%. Very surprising that cause of death such as infection, haemorrhage was not determined?

..., UV sterilization for 3 hours was conducted.
I consider this sterilization method to be inadequate for a material to be surgically implanted. Because UV light will only disinfect the surface and will not have an effect of the interior of the implant particles. Authors need to provide evidence about how this sterilization method was selected using general surgical principals.

Page 6: ... using a drill.
Give more details of the bone drilling process. What was the speed of the motor? The type and size of the bur? Was the bur irrigated during drilling?

fill the defect with green mussel shell HA.
What was the form of HA? Was it a powder? If so what is the size of the particles?

The blood sampling was conducted by inserting needle parallel to the lateral marginal auricular vein. Once the needle reached the vein, blood will flow into the catheter. The vacutainer tube was pushed so that blood can enter the 1-2 ml tube. After collecting the required sample, the needle was immediately removed from the rabbit’s lateral marginal vein. The injected area was pressed with an alcohol swab at the end of this process.
It is not necessary to provide minute details on venopuncture. It would have been preferable if this much of details were provided for the surgical technique of HA implantation.

Examination of ALP is selected due to its availability in most health centers.
Question is blood samples tested in Animal Health Centres are that of common companion animals such as dogs and cats. Therefore, can testing ALP in rabbit blood in a health centre be accurate?

Page 9: As a result, the presence of a hydroxyapatite scaffold reduces ALP activity since the body no longer needs to synthesize hydroxyapatite from PPi.
It can also be argued that the low ALP level indicate that the rate of bone remodelling is slow, leading to the rate of new bone formation to be also being low.

This experiment had a number of methodological limitations, including fractures in the rabbits and post-interventional infections that were not equally exposed to all of the rabbits. This may impact the trial outcomes by increasing the time it takes for bones to recover, which could prevent all treatment groups from showing significant results between the weeks 2, 4, and 6. Even though all groups received an appropriate dose of 300 mg at the area of the lesion, the prolonged bone
healing time meant that the test between the positive control group and the negative control group did not produce significant results in the week 6.
If most of the animals in this study experienced delayed healing of surgical sites due to infections, then the outcome of the complete investigation is brought into question?

..... negative control group (K1.1)
In the negative control, although there was no implant material in the surgical site, the bone defect would not have been empty, it would have been filled with blood clot that eventually got organised, leading to bone healing with new bone formation. Therefore, the significant difference between negative group and the treatment group may indicate that the green mussel shell hydroxyapatite may not be effective as the natural blood clot?

A comparison test of the serum ALP levels at the weeks 2, 4, and 6 in each treatment group’s results did not reveal any significant differences. This indicates that while ALP activity varied at weeks 2, 4, and 6, the differences were not statistically significant. This means that the effect of giving HA works evenly in the weeks 2, 4, and 6.
It is very curious why the authors did not take bone tissue biopsies from the surgical sites at these time points and investigate the histology, and compare these findings with that of the ALP values?

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?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biomaterials, in-vitro and in-vovo investigations.Implants for surgical applications.

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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[1] 1. Andrzejowski P, Giannoudis PV: The ‘diamond concept’ for long bone non-union management. J. Orthop. Traumatol. 2019; 20(1): 1–3.

[2] 2. Qamar N, Aswari A: Healing or Hurting: development of highway public transportation technology. J. Dinamika Huk. 2018; 18(3): 319–328.

[3] 3. Kementerian Kesehatan RI, Hasil Riset Kesehatan Dasar (Riskesdas): 2018. Badan Penelitian dan Pengembangan Kesehatan Kementerian RI.2018.

[4] 4. Amin S, Achenbach SJ, Atkinson EJ, et al.: Trends in fracture incidence: a population-based study over 20 years. J. Bone Miner. Res. 2014; 29(3): 581–589. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Noorisa R, Apriliwati D, Aziz A, et al.: The characteristic of patients with femoral fracture in Department Of Orthopaedic And Traumatology RSUD Dr. Soetomo Surabaya 2013–2016. J. Orthop. Traumatol. Surabaya. 2017; 6(1): 1–1.

[6] 6. Babcock S, Kellam JF: Hip fracture nonunions: diagnosis, treatment, and special considerations in elderly patients. Adv. Orthop. 2018; 2018: 1–11. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Frölke JP, Patka P: Definition and classification of fracture non-unions. Injury. 2007; 38: S19–S22. PubMed Abstract | Publisher Full Text

[8] 8. Kattimani VS, Kondaka S, Lingamaneni KP: Hydroxyapatite–-Past, present, and future in bone regeneration. Bone Tissue Regen. Insights. 2016; 7: BTRI.S36138–BTRI.S36119. Publisher Full Text

[9] 9. Bhatt RA, Rozental TD: Bone graft substitutes. Hand Clin. 2012; 28(4): 457–468. Publisher Full Text

[10] 10. Vecchio KS, Zhang X, Massie JB, et al.: Conversion of bulk seashells to biocompatible hydroxyapatite for bone implants. Acta Biomater. 2007; 3(6): 910–918. PubMed Abstract | Publisher Full Text

[11] 11. Dhanaraj K, Suresh G: Conversion of waste sea shell (Anadara granosa) into valuable nanohydroxyapatite (nHAp) for biomedical applications. Vacuum. 2018; 152: 222–230. Publisher Full Text

[12] 12. Sangwaranatee NW, Teanchai K, Kongsriprapan S, et al.: Characterization and analyzation of chitosan powder from Perna Viridis shell. Materials Today: Proceedings. 2018; 5(6): 13922–13925.

[13] 13. Fitriah E, Maryuningsih Y, Roviati E: Pemanfaatan daging dan cangkang kerang hijau (Perna viridis) sebagai bahan olahan pangan tinggi kalsium. Proceeding of The URECOL. 2018; pp. 412–423.

[14] 14. Liemawan AE, Tavio T, Raka IG: Pemanfaatan limbah kerang hijau (Perna Viridis L.) sebagai bahan campuran kadar optimum agregat halus pada beton mix design dengan metode substitusi. Jurnal Teknik ITS. 2015; 4(1): F128–F133.

[15] 15. Nazht HH, Omara RA, Al Dahhan MR, et al.: Estimation of alkaline phosphatase enzymes level in the femoral transverse fractures healing in rabbits.2019. Publisher Full Text

[16] 16. Rathwa HS, Verma T, Chavali VH: Assessment of union in fractures: Role of Serum Alkaline Phosphatase and Ultrasonography. J. Clin. Orthop. Trauma. 2021; 14: 94–100. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. Ismail R, Laroybafih MB, Fitriyana DF, et al.: The effect of hydrothermal holding time on the characterization of hydroxyapatite synthesized from green mussel shells. Journal of Advanced Research in Fluid Mechanics and Thermal Sciences. 2021; 80(1): 84–93. Publisher Full Text

[18] 18. Ajayi IE, Shawulu JC, Zachariya TS, et al.: Osteomorphometry of the bones of the thigh, crus and foot in the New Zealand white rabbit (Oryctolagus cuniculus). Ital. J. Anat. Embryol. 2012; 117(3): 125–134. PubMed Abstract

[19] 19. Bagi CM, Berryman E, Moalli MR: Comparative bone anatomy of commonly used laboratory animals: implications for drug discovery. Comp. Med. 2011; 61(1): 76–85. PubMed Abstract

[20] 20. Nazht HH: Estimation the alkaline phosphatase (ALP) level in partial hepatectomy rabbits. Al-Qadisiyah Journal of Veterinary Medicine Sciences. 2015; 14(1): 10–14.

[21] 21. Washington IM, Van Hoosier G: Clinical Biochemistry and Hematology. The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents. 2012; pp. 57–116. Publisher Full Text

[22] 22. Orimo H: The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J. Nippon Med. Sch. 2010; 77(1): 4–12. PubMed Abstract | Publisher Full Text

[23] 23. Stigbrand T, Wahren B: Alkaline Phosphatases. Serological Cancer. Markers. 1992; 11: 135. Publisher Full Text

[24] 24. Tjandra KC, Novriansyah R, Limijadi EKS, et al.: The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect. Mendeley Data. 2023; V1. Publisher Full Text

[25] 25. Tjandra KC, Novriansyah R, Limijadi EKS, et al.: The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect. Mendeley Data. 2023; V1. Publisher Full Text

The effect of green mussel (Perna viridis) shells’ hydroxyapatite application on alkaline phosphatase levels in rabbit femur bone defect

Abstract

Keywords

Introduction

Methods

Ethics

Design

Animals and materials

Figure 1. Study group design.

Table 1. Humane endpoint criteria.26

Procedure

Analysis

Results

Table 2. Rabbit serum alkaline phosphatase level (U/L).

Table 3. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the positive control group (bovine) (U/L).

Table 4. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the intervention group (green mussel shell) (U/L).

Table 5. One-way ANOVA test results for serum alkaline phosphatase levels in rabbits in the negative control group (U/L).

Table 6. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-2nd (U/L).

Table 7. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-4th (U/L).

Table 8. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6th (U/L).

Table 9. The results of the LSD post hoc test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6th (U/L).

Discussion

Conclusion

Data availability

Underlying data

Reporting guidelines

References

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Table 1. Humane endpoint criteria.²⁶

Table 6. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-2^nd (U/L).

Table 7. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-4^th (U/L).

Table 8. The results of the one-way ANOVA test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6^th (U/L).

Table 9. The results of the LSD post hoc test for serum alkaline phosphatase levels of positive control, treatment, and negative control groups at week-6^th (U/L).