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Pratiwi Soesilawati,1 Harianto Notopuro,2 Yuliati Yuliati,1 Maretaningtias Dwi Ariani,3 Muhammad Alwino Bayu Firdauzy41Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia; 2Department of Biochemistry, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia; 3Department of Prosthodontics, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia; 4Faculty of Dental Medicine, Universitas Airlangga, Surabaya, IndonesiaCorrespondence: Pratiwi SoesilawatiDepartment of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga, Jl. Prof. Dr. Moestopo No. 47 Surabaya, East Java, IndonesiaTel +62 31 503 0255Email pratiwi-s@fkg.unair.ac.idPurpose: The aim of this study is to asses the relationship between the level of sIgA and dental caries experience in healthy children who are 6- to 9-years-old from Indonesia. The case-control study is conducted to determine the protective role of salivary secretory immunoglobulin A (sIgA) levels in the stimulated whole saliva of dental caries-active and caries-free children.Methods: This research was done by stimulating the whole saliva which had been collected from 6- to 9-years-old children with the index def-t≥3 of 30 children as the caries-active children group and 30 children with def-t

Placenta accreta spectrum (PAS) is defined as abnormal placental adherence or invasion of the myometrium or extrauterine organs. This case series will analyze MRI findings and PAS grading, in addition to emergency situations like massive hematuria and placental invasion with rupture. We report 5 cases of pregnant women with placenta previa with suspected PAS. MRI revealed 1 case of placenta accreta, one case of placenta increta, and 3 cases of placenta percreta. Two cases were emergency situations. All cases were managed with cesarean section. PAS is frequently related to severe obstetric hemorrhage associated with high maternal morbidity and mortality, making diagnosis and management challenging. Ultrasound is the initial diagnostic modality for PAS. Although ultrasound is preferred for PAS diagnosis, MRI provides an effective modality for the analysis of the depth of placental invasion and can be helpful in emergency situations.

Introduction: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) or 11-hydroxylase deficiency (11OHD) is characterized by underproduction of cortisol and overproduction of adrenal androgens. These androgens lead to a variable degree of virilization of the female external genitalia in 46,XX individuals. Especially in developing countries, diagnosis is often delayed and 46,XX patients might be assigned as males. This study aims to describe the clinical and biochemical characteristics of a unique cohort of untreated male-reared 46,XX classic CAH patients from Indonesia and discusses treatment challenges., Methods: Nine untreated classic CAH patients with 46,XX genotype and 21OHD (n=6) or 11OHD (n=3), aged 3-46 years old, were included. Biometrical parameters, clinical characteristics, and biochemical measurements including glucocorticoids, renin, androgens, and the pituitary-gonadal axis were evaluated., Results: All patients had low early morning serum cortisol concentrations (median 89 nmol/L) without significant increase after ACTH stimulation. Three patients with salt wasting 21OHD reported one or more periods with seizures and/or vomiting in their past until the age of 6, but not thereafter. The remaining patients reported no severe illness or hospitalization episodes, despite their decreased capacity to produce cortisol. In the 21OHD patients, plasma renin levels were elevated compared to the reference range, and in 11OHD patients renin levels were in the low-normal range. All adult patients had serum testosterone concentrations within the normal male reference range. In 21OHD patients, serum 11-oxygenated androgens comprised 41-60% of the total serum androgen concentrations. Glucocorticoid treatment was offered to all patients, but they refused after counseling as this would reduce their endogenous androgen production and they did not report complaints of their low cortisol levels., Discussion: We describe a unique cohort of untreated classic 46,XX male CAH patients without overt clinical signs of cortisol deficiency despite their cortisol underproduction and incapacity to increase cortisol levels after ACTH stimulation. The described adolescent and adult patients produce androgen levels within or above the normal male reference range. Glucocorticoid treatment will lower these adrenal androgen concentrations. Therefore, in 46,XX CAH patients reared as males an individual treatment approach with careful counseling and clear instructions is needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Adriaansen, Utari, Westra, Juniarto, Ariani, Ediati, Schröder, Span, Sweep, Drop, Faradz, van Herwaarden and Claahsen – van der Grinten.)

Statement of Problem: Information regarding facial landmark measurement using machine learning (ML) techniques in prosthodontics is lacking., Purpose: The objective of this study was to evaluate and compare the reliability, validity, and accuracy of facial anthropological measurements using both manual and ML landmark detection techniques., Material and Methods: Two-dimensional (2D) frontal full-face photographs of 50 men and 50 women were made. The interpupillary width (IPW), interlateral canthus width (LCW), intermedial canthus width (MCW), interalar width (IAW), and intercommissural width (ICW) were measured on 2D digital images using manual and ML methods. The automated measurements were recorded using a programming language (Python), and a convolutional neural network (CNN) model was trained to detect human facial landmarks. The obtained data from the manual and ML methods were analyzed using intraclass correlation coefficients (ICCs), the paired sample t test, Bland-Altman plots, and the Pearson correlation analysis (α=.05)., Results: Intrarater and interrater reliability values were greater than 0.90, indicating excellent reliability. The mean difference between the manual and ML measurements of IPW, MCW, IAW, and ICW was 0.02 mm, while it was 0.01 mm for LCW. No statistically significant differences were found between the measurements obtained by the manual and ML methods (P>.05). Highly significant positive correlations (P<.001) were obtained between the results of the manual and ML methods: (r=0.996[IPW], r=0.977[LCW], r=0.944[MCW], r=0.965[IAW], and r=0.997[ICW])., Conclusions: In the field of prosthodontics, the use of ML methods provides a reliable alternative to manual digital techniques for carrying out facial anthropometric measurements., (Published by Elsevier Inc.)

Kaempferia galanga rhizome is traditionally used as a treatment for various diseases. Ethyl p -methoxycinnamate (EPMC), which constitutes up to 31.77% of the total essential oil, is the main/marker compound. EPMC is responsible for various pharmacological activities of Kaempferia galanga rhizome. According to the existing research, the isolation yield of EPMC is still meager, namely 0.50-2.50%; thus, a new EPMC isolation method is needed to produce better results. In this study, after determining the association constant and obtaining the Jobs plot between methacrylate derivative monomers and EPMC, a molecularly imprinted polymer for solid phase extraction (MI-SPE) was synthesized through bulk polymerization with EPMC as a template, methacrylic acid as a monomer, TRIM/EDGMA as a crosslinker in a ratio of 1 : 4 : 20 (MIP1) or 1 : 7 : 20 (MIP2). BPO was used as an initiator and n -hexane was used as a porogen. The synthesis of the NIP was also conducted using the same ratio but without the template. The MIPs were then characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and Brunauer-Emmett-Teller (BET) measurements, and their analytical performance was evaluated through adsorption capacity and selectivity. The results indicate that MIP2 exhibits better analytical performance with an adsorption capacity value of 0.0813 mg g -1 . The selectivity of MIP2 was tested using EPMC analog compounds, namely ethyl cinnamic (EC), cinnamaldehyde (CD), and kaempferol (KF), with imprinting factor (IF) values of 17.436, 1.539, and 0.06, respectively. Lastly, MIP2 was applied to the SPE cartridge for the isolation of EPMC from Kaempferia galanga rhizome extract, and showed a percentage recovery of 82.40% for the ethanol extract, 68.05% for the ethyl acetate extract, and 65.27% for the n -hexane extract. MI-SPE 2 gives high purity results for the ethanol, ethyl acetate, and n -hexane extracts, with purities of 97.00%, 97.63%, and 99.59%, respectively. These results indicate that the MI-SPE technique shows great potential as a new method for isolating EPMCs with high yield and purity., Competing Interests: There are no conflicts to be declare., (This journal is © The Royal Society of Chemistry.)

The membranes used in bone reconstructions have been the object of investigation in the field of tissue engineering, seeking to improve their mechanical strength and add other properties, mainly the osteopromotive. This study aimed to evaluate the functionalization of collagen membranes, with atomic layer deposition of TiO 2 on the bone repair of critical defects in rat calvaria and subcutaneous biocompatibility. A total of 39 male rats were randomized into four groups: blood clot (BC), collagen membrane (COL), COL 150-150 cycles of titania, and COL 600-600 cycles of titania. The defects were created in each calvaria (5 mm in diameter) and covered according to each group; the animals were euthanized at 7, 14, and 28 days. The collected samples were assessed by histometric (newly bone formed, soft tissue area, membrane area, and residual linear defect) and histologic (inflammatory cells and blood cells count) analysis. All data were subjected to statistical analysis ( p < 0.05). The COL150 group showed statistically significant differences compared to the other groups, mainly in the analysis of residual linear defects (1.5 ± 0.5 × 10 6 pixels/µm 2 for COL 150, and around 1 ± 0.5 × 10 6 pixels/µm 2 for the other groups) and newly formed bone (1500 ± 1200 pixels/µm for COL 150, and around 4000 pixels/µm for the others) ( p < 0.05), demonstrating a better biological behavior in the chronology of defects repair. It is concluded that the collagen membrane functionalized by TiO 2 over 150 cycles showed better bioactive potential in treating critical size defects in the rats' calvaria.

Background: Our multidisciplinary team (MDT) is a large specialized team based in Semarang, Indonesia, that cares for a wide variety of pediatric and adult individuals with differences of sex development (DSD) from across Indonesia. Here, we describe our work over the last 17 years., Methods: We analyzed phenotypic, hormonal, and genetic findings from clinical records for all patients referred to our MDT during the period 2004-2020., Results: Among 1,184 DSD patients, 10% had sex chromosome DSD, 67% had 46,XY DSD, and 23% had 46,XX DSD. The most common sex chromosome anomaly was Turner syndrome (45,X) (55 cases). For patients with 46,XY DSD under-masculinization was the most common diagnosis (311 cases), and for 46,XX DSD, a defect of Müllerian development was most common (131 cases) followed by congenital adrenal hyperplasia (CAH) (116 cases). Sanger sequencing, MLPA, and targeted gene sequencing of 257 patients with 46,XY DSD found likely causative variants in 21% (55 cases), with 13 diagnostic genes implicated. The most affected gene codes for the androgen receptor. Molecular analysis identified a diagnosis for 69 of 116 patients with CAH, with 62 carrying variants in CYP21A2 including four novel variants, and 7 patients carrying variants in CYP11B1. In many cases, these genetic diagnoses influenced the clinical management of patients and their families., Conclusions: Our work has highlighted the occurrence of different DSDs in Indonesia. By applying sequencing technologies as part of our clinical care, we have delivered a number of genetic diagnoses and identified novel pathogenic variants in some genes, which may be clinically specific to Indonesia. Genetics can inform many aspects of DSD clinical management, and while many of our patients remain undiagnosed, we hope that future testing may provide answers for even more., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Macroadenoma is a tumor that typically develops in the epithelial cells of the pituitary gland. Patients suffering from the condition are often asymptomatic with complaints that are caused by hormonal imbalance. Therefore, chromosome analysis needs to be done to females aged >16 years presenting with amenorrhea. Karyotype 46,XY is a disorder of sex development (DSD) that is caused by the complex process of gene interactions, androgen synthesis, and hormone regulation. The patient initially came to the hospital for a scheduled transsphenoidal surgery due to pituitary macroadenoma, and later complained of primary amenorrhea and atypical external genital. Furthermore, physical examination of genitalia revealed mild clitoromegaly without obvious introitus vagina. Laboratory testing showed elevated prolactin and testosterone level, while ultrasonography imaging revealed the absence of the uterus and ovaries. The brain magnetic resonance imaging (MRI) demonstrated a pituitary adenoma, and cytogenetic analysis showed 46,XY karyotype. Subsequently, hyperprolactinemia, imaging, and histopathology examination were used to confirm pituitary macroadenoma in the patient. It was assumed that the undermasculinized genitalia was caused by hormonal disorders including the deficiency of androgen action or 5-alpha-reductase enzyme. 46,XY DSD has many different symptoms, hence, clinicians need to be aware of potential multifactorial aetiologies. Imaging of internal genitalia, hormonal and chromosomal analysis should be carried out to assess patients with unknown causes of the disorder. Molecular analysis needs to be carried to exclude the possible gene mutation.

Background: Congenital Adrenal Hyperplasia (CAH) due to CYP11B1 is a rare autosomal recessive adrenal disorder that causes a decrease in cortisol production and accumulation of adrenal androgens and steroid precursors with mineralocorticoid activity. Clinical manifestations include cortisol deficiency, ambiguous genitalia in females (differences of sex development (DSD)), and hypertension. Medical treatment recommendations are well defined, consisting of glucocorticoid treatment to substitute glucocorticoid deficiency and consequently normalize adrenal androgen and precursors levels. Current guidelines also emphasize the need for specialized multidisciplinary DSD teams and psychosocial support. In many developing countries, care for DSD patients, especially when caused by an adrenal disease, is challenging due to the lack of infrastructure, knowledge, and medication., Objective: The study aims to report the conflicting decision-making process of medical treatment and sex assignment in late-identified CAH patients in developing countries., Methods: We describe the clinical and biochemical findings and the psychological assessment of five affected but untreated family members with CAH due to CYP11B1 deficiency., Results: All patients had a 46,XX karyotype, ambiguous genitalia, low cortisol levels, and hypertension. Two identified as males, two as females, and one had undecided gender. The patients were counselled that refusing treatment will lead to infertility and the potential risk of developing Addisonian crisis and severe hypertension. However, all 46,XX CAH males refused treatment with glucocorticoids due to the expected lowering of adrenal androgens as their main source of testosterone. None of the patients developed Addisonian crisis, probably due to some residual cortisol activity and glucocorticoid activity of elevated adrenal steroid precursors., Conclusion: Medical treatment and sex assignment in late-identified 46,XX CAH patients in Indonesia may often depend on local and cultural factors. The management of DSD conditions may have to be individualized and integrated into the psychological and social context of the affected family., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Utari, Faradz, Ediati, Rinne, Ariani, Juniarto, Drop, van Herwaarden and Claahsen-van der Grinten.)

During the last few years, separation techniques using molecularly imprinted polymers (MIPs) have been developed, making breakthroughs using magnetic properties. Compared to conventional MIPs, magnetic molecularly imprinted polymers (MMIPs) have advantages in sample pretreatment due to their high specificity and selectivity towards analytes as a result of their larger specific surface areas and highly accessible specific binding sites. The techniques of isolation of active compounds from natural products usually require very long process times and low compound yields. When MMIPs are used in sample separation as Solid Phase Extraction (SPE) sorbents, the MMIPs are introduced into the dissolved sample and spread evenly, and they form bonds between the analyte and the MMIPs, which are then separated from the sample matrix using an external magnetic field. This process of separating analytes from the sample matrix makes the separation technique with MMIPs very simple and easy. This review discusses how to synthesize MMIPs, which factors must be considered in their synthesis, and their application in the separation of active compounds from natural products. MMIPs with magnetic core-shells made by co-precipitation can be a good choice for further development due to the high synthesis yield. Further optimization of the factors affecting the size and distribution of magnetic core-shell particles can obtain higher synthesis yields of MMIPs with higher adsorption capacity and selectivity. Thus, they can isolate target compounds from natural plants in high yields and purity.

Objective: There is still a great deal to learn about the influence of sex in systemic sclerosis (SSc). In this respect, national registries provide large and homogeneous patient cohorts for analytical studies. We therefore investigated a wide-ranging and well-characterized SSc series with the aim of identifying sex differences in disease expression, with a special focus on demographic, clinical, and serological characteristics., Methods: A multicenter SSc cohort of 2281 patients, including 247 men, was recruited in the Italian Systemic sclerosis PRogression INvestiGation (SPRING) registry. Demographic data, disease manifestations, serological profile, and internal organ involvement were compared., Results: The overall female/male ratio was 8.2:1. Female/male ratios for limited cutaneous SSc, diffuse cutaneous SSc, and SSc sine scleroderma subsets were 8.7:1, 4.9:1, and 10.7:1, respectively. A shorter time from onset of Raynaud phenomenon to SSc diagnosis, an increased prevalence of the diffuse cutaneous subset, renal crisis, and digital ulcers were found in males, whereas a significantly higher percentage of sicca syndrome, serum antinuclear antibodies, antiextractable nuclear antigens, anti-La/SSB, and anticentromere protein B was detected in the female group. Males exhibited lower left ventricular ejection fraction, as well as higher prevalence of conduction blocks, arrhythmias, ground glass, and honeycombing. Moreover, forced vital capacity and total lung capacity were medially lower in men than in women. Finally, males were more frequently treated with immunosuppressive drugs., Conclusion: Our study further supports the presence of several sex-related differences in patients with SSc. These differences were pronounced in the severity of cutaneous, peripheral vascular, and cardiopulmonary involvement for male patients, whereas an increased prevalence of sicca syndrome and a specific autoantibody profile characterized the female sex., (© 2022 by the Journal of Rheumatology.)

Objective:  This study aims to combine natural propolis with bovine bone graft (BBG) as a means of extraction socket preservation after 3 and 7 days toward expression of heat shock protein (HSP) 70 and osteocalcin to regenerate bone., Materials and Methods:  The Cavia cobaya were divided into eight groups, each consisting of seven samples. Their lower left incisors were extracted and induced with PEG, propolis extract, BBG, and a combination of propolis extract BBG. The research subjects were terminated on days 3 and 7 postextraction. Immunohistochemical and histopathological examinations were subsequently performed to observe HSP 70 expression, osteocalcin expression, osteoblasts, and osteoclasts., Statistical Analysis:  Data obtained were then analyzed with one-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) tests., Results:  Both the groups with the combination of propolis extract and BBG on days 3 and 7 were found to present the highest number of HSP70 expression, osteocalcin expression, and osteoblast cells as well as the lowest number of osteoclasts., Conclusion:  Both the groups with the combination of propolis extract and BBG on days 3 and 7 were found to present the highest number of HSP70 expression, osteocalcin expression, and osteoblast cells as well as the lowest number of osteoclasts., Competing Interests: None declared.

Background: Low levels of estrogen can cause osteoporosis and usually occur during a woman's menopausal phase. Osteoporosis can lead to bone resorption, the absence of osseointegration, and implant failure. The aim of this study is to determine the expression of transforming growth factor-beta 1 (TGF-β1), runt-related transcription factor (RUNX2), and osteoblasts in mandibular rats with low levels of estrogen., Materials and Methods: This study is an in vivo experimental research. Female Wistar rats ( n = 18) were divided into two groups: (1) Postsham surgery and (2) ovariectomy group. After 12 weeks, the rats were sacrificed to identify the level of estrogen, while histological analysis was conducted to determine the level of osteoblast and the expression of TGF-β1 and RUNX2. The data were analyzed using t -test ( P < 0.05)., Results: There were significant lower levels of estrogen and osteoblast among the ovariectomy group compared to the postsham group ( P < 0.05). RUNX2 levels were found to be significantly higher in the ovariectomy group than that in the postsham group ( P < 0.05). However, there were no significant differences between TGF-β1 levels within the ovariectomy and postsham groups ( P > 0.05)., Conclusion: Ovariectomy can lead to decreased osteoblastogenesis in mandibular bone by the reduced level of osteoblast and the increased expression of TGF-β1 and RUNX2., Competing Interests: The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial, in this article.

Objectives: To describe the phenotype variation in Indonesian 46,XX late-identified congenital adrenal hyperplasia (CAH) and the correlation between 17-hydroxyprogesterone (17-OHP) and genital virilization., Methodology: Retrospective study of 39 cases with five salt-wasting (SW) and 34 simple virilizing (SV) types., Results: The median age of the patients was 9.83 years (range, 0.58 to 44 years) with Prader score 2 to 5. Clitoromegaly (100%) and skin hyperpigmentation (87%) were the most common features. Lack of breast development (Tanner 1 to 2) and menstrual disorders occurred in 9 patients (teenagers and adults). Short stature (6), low voice (14), prominent Adam's apple (9) and hirsutism (4) were found only in SV types. Rapid growth (7) and precocious puberty (8) were identified in children. Male gender on admission was found in 13 patients. The mean of 17-OHP level was 304.23 nmol/L [standard deviation (SD) 125.03 nmol/L]. There was no correlation between 17-OHP levels and virilization (r=0.19, p>0.05)., Conclusion: Late-identified CAH showed severe virilization and irreversible sequelae, with clitoromegaly and skin hyperpigmentation as the most commonly seen features. Masculinization of CAH females created uncertainty with regard to sex assignment at birth, resulting in female, male and undecided genders. There is no significant correlation between 17-OHP levels with the degree of virilization in CAH females., Competing Interests: The authors declared no conflict of interest., (© 2018 Journal of the ASEAN Federation of Endocrine Societies.)

Background: Following the extraction of a tooth, bone resorption can cause significant problems for a subsequent denture implant and restorative dentistry. Thus, the tooth extraction socket needs to be maintained to reduce the chance of any alveolar ridge bone resorption., Objective: The objective of this study is to determine whether the administration of mangosteen peel extracts (MPEs), combined with demineralized freeze-dried bovine bone xenograft (DFBBX) materials for tooth extraction socket preservation, could potentially reduce inflammation by decreased the expression of nuclear factor κβ (NfKb) and receptor activator of nuclear factor-κβ ligand (RANKL), to inhibit alveolar bone resorption, and increased of bone morphogenetic protein-2 (BMP2) expressions to accelerate alveolar bone regeneration., Materials and Methods: This study consists of several stages. First, a dosage of MPE combined with graft materials was applied to a preserved tooth extraction socket of a Cavia cobaya . Second, the C. cobaya was examined using immune histochemical expression of NfKb, RANKL, BMP2, as well as histology of osteoblasts and osteoclasts. The research was statistically analyzed, using an analysis of variance test and Tukey honest significant difference test., Results: The results of this research were that it was determined that MPEs combined with graft materials on a preserved tooth extraction socket can reduce NfKb, RANK, and osteoclasts also increase of BMP2 and osteoblast., Conclusion: The induction of MPEs and DFBBX is effective in reducing inflammation, lowering osteoclasts, decreasing alveolar bone resorption, and also increasing BMP2 expression and alveolar bone regeneration., Competing Interests: There are no conflicts of interest.

Objective: The objective of this study was to determine the aetiological spectrum of disorders of sex development (DSD) in a large cohort of underprivileged and undiagnosed patients from Indonesia., Methods: A total of 286 patients with atypical external and/or internal genitalia were evaluated using clinical, hormonal, molecular genetic and histological parameters., Results: The age (years) at presentation was 0-0·5 in 41 (14·3%), >0·5-12 in 181 (63·3%) and >12 in 64 cases (22·4%). 46,XY DSD was most common (68·2%, n = 195), 46,XX DSD was found in 23·4% (n = 67) and sex chromosomal DSD in 8·4% (n = 24). In 61·2% of 46,XX DSD patients, 17·9% of 46,XY DSD patients and all sex chromosome DSD patients (29·4% in total), a final diagnosis was reached based on genetic or histological gonadal tissue evaluation. 17-hydroxyprogesterone and androstenedione levels were the most distinctive parameters in 46,XX DSD patients. In 46,XY DSD, diagnostic groups were identified based on the external masculinization score: androgen action disorder (AAD), unknown male undermasculinization (UMU), and gonadal dysgenesis (GD). LH, FSH and testosterone levels were most informative especially in the older age group. HCG tests were of no additional value as no patients with androgen synthesis disorders were found. Hormonal profiles of patients with sex chromosome DSD and a Y-chromosome sequence containing karyotype showed high levels of LH and FSH, and low levels of AMH, inhibin B and testosterone compared with the normal male range. Gene mutations were found in all patients with CAH, but in only 24·5% and 1·8% of patients with AAD and UMU. In 32% of 46,XY GD patients, copy number variants of different genes were found., Conclusion: A stepwise diagnostic approach led to a molecularly or histologically proven final diagnosis in 29·4% of the patients. The most informative parameters were serum levels of 17-hydroxyprogesterone and androstenedione in 46,XX DSD patients, and serum LH, FSH and testosterone levels in 46,XY DSD patients., (© 2016 John Wiley & Sons Ltd.)

Background: Collagen is regarded as one of the most useful biomaterials. We tried to combine collagen and carbonate apatite (CA) with some cytokines in order to enhance bone formation ability. In this study, we found that CA-collagen sponge (CA-CS) was a possible candidate of newly graft material for bone formation., Materials and Methods: CA-CS was fabricated by the following procedure. One wt% of pig hide collagen solution (Nippon Meat Packers. Inc., Tokyo, Japan) was neutralized with 0.1 N NaOH, and then mixed immediately 243 mg apatite powder with 0.06 M carbonate contents. After centrifugation at 1500 rpm for 10 min, excess water was removed, and the mixture was packed into Teflon molds (5.0 mm × 2.0 mm). Each 10 µg of basic fibroblast growth factor (bFGF) and recombinant human bone morphogenetic protein-2 (rh-BMP2) were involved in these sponges. Then these scaffolds frozen at -80°C for 2 h and dried in a freeze dry machine for 24 h. CA-CS without cytokines were also prepared as a control. Mouse osteoblast-like cell (MC3T3-E1) proliferations in these scaffolds were investigated by 3-day in vitro cell culture using MTT assay examination. Ten New Zealand rabbits (weight: 3-3.5 kg) were used in this in vivo study. After 3 weeks of placement, the scaffolds, rabbits were sacrificed, and bone formation in the sockets was evaluated histologically and histomorphometrically., Results and Conclusion: By histological observation and measurement of bone area ratio, CA-CS with cytokines showed higher bone formation ability (bFGF/CA-CS: 50.7 ± 7.3%, rh-BMP2/CA-CS: 54.2 ± 5.0%) than other groups. From the limited results of this study, it is suggested that CA collagen scaffolds with some cytokines may become an attractive scaffold for bone regeneration.

Carbonate apatite-chitosan scaffolds (CA-ChSs) were fabricated using the lyophilization technique. It was found that ChSs prepared with 200 mg chitosan powder (ChSs200) had well-structured three-dimensional architecture with high porosity and good retentive form without brittleness. In addition, it was shown that the number of osteoblast-like cells MC3T3-E1 proliferated on desalinated ChSs200 was larger than that on the non-desalinated ChSs200. CA-ChSs were fabricated by adding 100 mg carbonate apatite (CA) to 200 mg chitosan gels followed by freeze-drying (CA100ChSs200). SEM observation revealed that CA100ChSs200 had favorable three- dimensional porous structures. The number of living cells increased more rapidly on CA100ChSs200 prepared with different amounts of CA than on ChSs. ALP activity significantly increased after day 14 and reached a plateau after day 21 in ChSs200 and CA100ChSs200. It was concluded that newly developed CA100ChSs200 may be a possible scaffold material for tissue engineering.