The healing effect of cuttlefish bone on fractured bone in rat model

Background: Fractured bone healing requires three to nine months, which prolongs the patients’ morbidity. Long bone fracture is considered to be difficult due to the dependence of bodily mobility and freedom. Bone tissue engineering materials includes hydroxyapatite (HAp), titanium, alumina, and polymers. HAp is found to be heavily abundant in cuttlefish. This study is conducted to prove that the cuttlebone has an effect of accelerating the fractured long bone healing process. Methods: This is an experimental study using a total of 32 samples of Rattus norwegicus. The Treatment Group received the cuttlebone extract + 0.9% NaCl while the Control Group received only NaCL 0.9%. Both groups were fractured beforehand. On the 14 day the fractured area was harvested and assigned for histopathology and radiographic


INTRODUCTION
Cancer is still a health problem in the world especially in developing countries including Indonesia. One of these cancers is cervical cancer, whose incidence is still high and tends to increase. Cervical cancer ranks the second most common cancer after breast cancer, which is one of the main causes of death in women. 1,2, 3 High risk oncogenic human papillomavirus (HPV) play a causative role in the development of cervical cancer. 3,4,5, 6 Most sexually-active women are exposed to HPV infection during their lifetimes. To date, more than 120 HPV types have been found, of which more than 40 types of HPV infect the anogenital and other mucosal body, of these, 13-18 types belonging to high-risk type. 7 Approximately 90-95% of cervical cancer are caused by high-risk HPV-16 and 18. 3, 8 Early detection of this high-risk human papillomavirus infection is very important to prevent the development of cervical lesions into precancerous lesions and cancer. A study by Kose and Murat (2014) found about 15% of women infected with HPV develop cervical intraepithelial neoplasia (CIN) within 7 years. Invasive cancer developed at a rate of 1-3% after this infection, and the required period is approximately 25-40 years or a few months to several years depending on the precancerous lesion grade. 7,9 These infections provide cytopathic effect i.e. changes in cervical squamous epithelial cells that are a pathognomonic sign for productive HPV infection, which can be seen in both routine cytological screening with the Papanicolaou (Pap) test and histopathologic examination. 7, 10 The cytopathic effect called koilocytotic atypia, characterized by the presence of nuclear atypia i.e. the size of the nucleus varies and enlarging up to three times the normal nucleus size, hyperchromatic nucleus, irregular nuclear membrane, cavitation or cytoplasmic halo around the nucleus, and cell membrane thickening. 4,10,11, 12 The genesis of the cytoplasmic vacuole has remained unclear, particularly because both HPV DNA replication and virion assembly occur exclusively in the nucleus.

ORIGINAL ARTICLE
Molecular-based HPV testing such as Polymerase Chain Reaction (PCR) is not yet possible used as a screening program. An easier, cheaper and simpler method of examination is needed to detect earlier infection, which can be applied in developing countries with large populations, but still, has a sensitivity and specificity close to gold standard. 13 This study aims to determine the accuracy of histopathologic examination compared with PCR to diagnose HPV infection in the uterine cervix.

Specimen collection
This study design was a cross-sectional diagnostic test that was conducted during the years 2013-2014. The materials of this study were the paraffin-embedded tissue biopsy, or surgery of patients with clinically diagnosed have a cervical lesion at Obstetric and Gynecologic Department Faculty of Medicine Udayana University/ Sanglah General Hospital. Samples were collected on a consecutive basis until the required sample size was met. The conventional histopathology examination was as the predictor variable and PCR examination as the outcome variable.

Histopathology Examination and HPV DNA Detection
The specimen was processed and then stained with routine Hematoxylin & Eosin. Histopathologic examination was performed to determine the presence of cytopathic effects as a sign of suspicion of HPV infection. For detection of HPV DNA, the paraffin-embedded tissues from biopsy or surgery of patients that have been examined histopathologically, either with or without cytopathic effect were sent to Molecular Biology Laboratory Unit, Faculty of Medicine Udayana University. SPF10 primers at several dilutions were used to amplify the DNA. A total of 39 cervical lesion patients were included in this study.

Statistical Analysis
A descriptive characteristic of the data subject was tabulated. A 2×2 table was created to calculate the sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). Table 1, and the results of histopathologic and PCR examination display in Table 2 and Table 3. The positive cytopathic effect with conventional histopathologic examination show in Figure 1

ORIGINAL ARTICLE
positive result of DNA HPV with PCR examination shown in Figure 2.
A total of 39 cervical lesion patients were included in this study. Of these patients, the most distribution was obtained in the 31-40 and 41-50 year age group of 12 samples (30.77%) each. Table 2 show the number of cases with positive cytopathic effect as a suspicion of HPV infection with the conventional histopathologic examination as many as 23 samples (58.97%), while with PCR were 22 samples (56.41%). The most prevalence of positive patients with conventional histopathologic and PCR examination was in the 31-40 years age group, each of 7 samples (17.95%) and 8 samples (20.51%). Table 3 show the accuracy of conventional histopathologic examination compared with PCR namely: sensitivity (Se) = 72.73%, specificity (Sp) = 58.82%, positive predictive value (PPV) = 69.57% and negative predictive value (NPV) = 62.50%.

DISCUSSION
Of the 39 samples studied, the most distribution of cervical, uterine lesion either with or without suspicion for HPV infection diagnosed with conventional histopathology was obtained in the 31-40 and 41-50 year age group of 12 samples (30.77%) each. This study is in accordance with the result of previous studies. A study by Ting et al. (2010) found age range of low-grade lesions occurred at 20-30 years age groups. 14 A cross-sectional study in Indonesia to evaluate the performance of a singlevisit approach of cervical cancer screening, using visual inspection with acetic acid (VIA), histology and cryotherapy in low-resource settings, found 64.4% high-risk women were in the age group of 30-49 years. 15 Young sexually active women are the highest risk for acquiring HPV infection. 7 Examination with conventional histopathology to determine the positive cytopathic effect as an indication of suspicion HPV infection was found in 23 samples (58.97%). Examination with PCR for positive DNA HPV was found in 22 samples (56.41%). The most prevalence of positive patients with conventional histopathology and PCR examination was in the 31-40 years age group, each of 7 samples (17.95%) and 8 samples (20.51%). All of the positive HPV samples in this study were low-grade precancerous lesions i.e. cervical intraepithelial neoplasia (CIN) grade 1. This result showed a higher percentage compared to the previous study i.e. the prevalence of HPV infection in precancerous lesion was 18% in CIN 1 and 66% in CIN 2-3. 11, 16 Studi by Hu et al. (2011) found 3.4% of their samples were diagnosed as CIN grade 2 or worse lesions, and 17.1% were found to be positive for HPV DNA. 17 In cervical cancer cases, infection by HPV found by 90-95% in the world, 18 and 95.9% in Jakarta. 19 A study by Vet et al. (2008) in three regions in Indonesia; Jakarta, Tasikmalaya, and Bali. Investigate age-specific prevalence of HPV types in a population-based sample of 2686 women, aged 15-70 years, found the overall HPV prevalence was 11.4%, and 92.9% was positive in cervical cancer patients. 20