BIOMIMETIC BIOREACTORS AS A TOOL FOR MORE RELEVANT BIOMATERIAL ASSESSMENT

Development of novel biomaterials for use in biomedical applications requires careful assessment due to the intended interactions with cells and tissues. Understanding biocompatibility, non-toxicity, and capability of promoting desired biological responses requires thorough characterization of biomaterial, including its chemical composition, surface properties, mechanical strength, degradation rate, etc. Traditional in vitro methods for evaluating biomaterials in cell monolayers are convenient but limited by the lack of specific biophysical signals found in vivo, which can lead to unreliable results. This in vitro-in vivo gap can result in the unnecessary sacrifice of a large number of animals for testing purposes. Therefore, there is a need for alternative approaches that beter mimic the in vivo environment and accurately predict the behavior of the biomaterial after implantation. Biomimetic bioreactors are primarily developed for tissue engineering to provide the key biochemical (e.g., nutrients, gases, growth factors) and biophysical signals (e.g., shear stress, hydrostatic pressure, mechanical strains) found in vivo and thus could be indispensable tools in physiologically relevant biomaterial assessment. Our group introduced the application of two biomimetic bioreactors for the physiologically relevant characterization of two types of composite biomaterials aimed for bone and osteochondral tissue engineering. In specific, macroporous composite scaffolds were produced using two natural polymers (gellan gum and alginate) as matrices imitating organic phase of bone tissue with incorporated particulate bioactive glass (BAG) and β-tricalcium phosphate (β-TCP) as hydroxyapatite (HAp) precursors. In addition, in osteochondral scaffolds, gellan gum hydrogel served as a cartilaginous layer on top of the porous composite base. Integrity and mechanical properties of all prepared scaffolds were monitored for 14 days under physiological levels of mechanical compression (up to 10% strain, compression rate 337.5 µm s-1) in a bioreactor with dynamic compression and medium perfusion. Bioactivity and HAp formation within the scaffolds were investigated in a perfusion bioreactor under the flow of simulated body fluid for up to 28 days. The scaffolds were assessed by SEM, EDS, and XRD analyses indicating a significant increase in HAp formation under bioreactor conditions as compared to static controls in all investigated samples. Moreover, the formed HAp crystals were more uniformly distributed throughout the scaffolds showing a more cauliflower-like morphology and thus, indicating potentials for bone/osteochondral tissue engineering applications. The obtained results confirm the high influence of experimental conditions on the outcomes of biomaterial characterization and importance of closely mimicking physiological conditions, thus puting forward biomimetic bioreactors as a means in this direction.Conference Abstracts / 33rd Annual Conference of the European Society for Biomaterials (ESB), 4-8 September, Davos, Switzerlan

Orofacijalna bol: dijagnostički i terapijski izazovi

The concept of diagnostics and therapy of musculoskeletal and neuropathic diseases of the stomatognathic system, which are the subject of this paper, has been developing for decades. It can be said that in order to avoid misunderstanding, the orofacial pain as a clinical problem, in the narrower sense, involves non-odontogenic and non-malignant causes of orofacial region. In this study, the results of clinical diagnosis of the population of 557 consecutive patients with orofacial pain based on multidisciplinary diagnostics were evaluated. 15.6% of patients have given up on the participation in the study. It has been shown that the patients who dropped out of the study were significantly older (p=0.0411) than those who agreed to participate, but there was no difference in gender ratio (p=0.185) since the proportion of female patients prevailed. In an analysis of 84.4% of patients participating in the study, the elevated anxiety values were established (mean value on STAI 1 was 39.2 and STAI 2 was 41.1) and statistical significance was found in correlation between elevated anxiety and intensity of pain as shown on visual analogue scale on open mouth (p<0.0001). Compared to the age, the statistical significance was for STAI 1 (p=0.0097) but not for STAI 2 (p=0.5599). The most common form of therapy is Michigan stabilization splint: for disc displacement of temporomandibular joint (TMJ) in 38.9% of patients and in combination with physiotherapy in 18.7% of patients; for osteoarthritis of TMJ in 28.4% and in combination with physiotherapy in 26.4% of patients. The treatment with anticonvulsant drugs for trigeminal neuralgia predominates in 54.3% of patients, which is combined with acupuncture in 25.7% of patients and only acupuncture in 17.1% of patients. In this study, a multidisciplinary co-operation in initial diagnostics and differential was designed to develop subspecialist knowledge on orofacial pain.Već desetljećima se razvija koncepcija dijagnostike i terapije muskuloskeletalnih i neuropatskih bolesti stomatognatskog sustava, što je predmet ovog članka. Kako bi se izbjegle nedoumice može se reći da orofacijalni bolovi kao klinički problem obuhvaćaju u užem smislu ne-odontogene i ne-maligne uzroke bolova orofacijalne regije. U ovom članku evaluirani su rezultati kliničke dijagnostike populacije od 557 konsekutivnih pacijenata s orofacijalnim bolovima temeljem multidisciplinarne dijagnostike. Od sudjelovanja u studiji odustalo je 15,6% pacijenata. Za pacijente koji su odustali od istraživanja pokazalo se da su bili značajnije stariji (p=0,0411) od onih koji su pristali sudjelovati, ali nije bilo razlike u omjeru spola (p=0,185), jer je prevladavao udio ženskih pacijenata. U analizi 84,4% pacijenata koji su sudjelovali u studiji utvrđene su povišene vrijednosti anksioznosti (prosjek STAI 1 je bio 39,2, a na STAI 2 je bio 41,1), te se je pokazala statistička značajnost u ovisnosti povišene anksioznosti s intenzitetom bolova na vizualno-analognoj skali pri otvorenim ustima (p<0,0001). U odnosu na dob statistička značajnost bilo je za STAI 1 (p=0,0097) ali ne i za STAI 2 (p=0,5599). Najčešći oblik terapije je michiganska stabilizacijska udlaga: za pomak diska temporomandibularnog zgloba (TMZ) kod 38,9% i u kombinaciji s fizioterapijom kod 18,7% pacijenata; za osteoartritis TMZ-a kod 28,4% i u kombinaciji sa fizioterapijom kod 26,4% pacijenata. Za trigeminalnu neuralgiju prevladava kod 54,3% liječenje sa antikonvulzivima, u kombinaciji s akupunkturom 25,7% te samo akupunktura kod 17,1% pacijenata. Multidisciplinarna suradnja u inicijalnoj dijagnostici i diferencijalnoj dijagnostici u ovoj studiji osmišljena je u svrhu razvijanja subspecijalističkog znanja o orofacijalnim bolovima

Asimetrija mandibule u pacijenata s unilateralnim pomakom diska temporomandibularnoga zgloba potvrđenog magnetskom rezonancijom

Objectives: The study aimed to determine the relationship between the mandibular asymmetry index according to Kjellberg between patients with painful unilateral anterior disc displacement (DD) and asymptomatic volunteers without disc displacement. Vertical measurements were performed on a panoramic single-image radiograph, and the disc status was confirmed by magnetic resonance imaging (MRI). Material and methods: Two groups of subjects were retrospectively selected, 40 patients (the overall mean age was 35.5 years; 75% female) with temporomandibular disorder symptoms confirmed by RDC/TMD axis I and manual functional analysis. Unilateral DD was determined by MRI. A comparative group of asymptomatic volunteers (20 dental students with a mean age of 23.4 years; 72% female) had the physiological position of the disc determined by MRI. The vertical asymmetry of the condyle was determined by the method of Kjellberg et al. The symmetry of the gonial angle of the mandible was also measured. Results: A comparison of the mean of the asymmetry index between patients (average 90.89±7.08%) and asymptomatic volunteers (mean 95.86±4.44%) showed a statistically significant difference (p=0.0029). There was no difference (p=0.088) in gonial angle symmetry between the patients (mean 96.48±2.96°) and the asymptomatic volunteers (mean 97.52±2.31°). The distribution of the presence of individual DD diagnoses (partial and total displacement with reduction, displacement without reduction) in patients diagnosed with asymmetry of the mandible was without statistical significance (p>0.05). Conclusion: This study actually points to the asymmetry of the mandible as a potential morphological risk of anterior DD.Svrha istraživanja: Cilj istraživanja bio je utvrditi povezanost indeksa asimetrije mandibule prema Kjellbergu između pacijenata s bolnim unilateralnim anteriornim pomakom diska i asimptomatskih dobrovoljaca bez pomaka diska. Vertikalna mjerenja obavljena su na ortopatomogramima, a status diska potvrđen je magnetskom rezonancijom. Materijal i metode: Retrospektivno su odabrane dvije skupine ispitanika od 40 pacijenata (prosječna dob 35,5 godina; 75 % ženskoga spola) sa simptomima temporomandibularnog poremećaja potvrđenima RDC/TMD-om os I i manualnom funkcijskom analizom. Unilateralni pomak diska ustanovljen je magnetskom rezonancijom. Usporednoj skupini asimptomatskih dobrovoljaca (20 studenata stomatologije prosječne dobi 23,4 godine; 72 % ženskoga spola) utvrđen je fiziološki položaj diska magnetskom rezonancijom. Vertikalna asimetrija kondila određena je metodom prema Kjellbergu i suradnicima. Mjerena je i simetrija gonijalnog kuta mandibule. Rezultati: Usporedba prosjeka indeksa asimetrije između pacijenata (prosjek 90,89 ± 7,08 %) i asimptomatskih dobrovoljaca (prosjek 95,86 ± 4,44 %) pokazala je statistički značajnu razliku (p = 0,0029). Nije bilo razlike (p = 0,088) u simetriji gonijalnog kuta između pacijenata (prosječno 96,48 ± 2,96°) i asimptomatskih dobrovoljaca (prosječno 97,52 ± 2,31°). Bez statističke značajnosti (p > 0,05) bila je distribucija prisutnosti pojedinih dijagnoza pomaka diska (djelomični i potpuni pomak s redukcijom, pomak bez redukcije) kod pacijenata kojima je utvrđena asimetrije mandibule. Zaključak: Ova studija dopušta povezivati asimetriju manidbule kao potencijalni morfološki rizik za nastanak anteriornoga pomaka diska

Učestalost pseudoeksfolijativnog sindroma i pseudoeksfolijativnog glaukoma u populaciji sjeverozapadne hrvatske u dobi od 40 godina i više

The purpose of this study was to assess the prevalence of pseudoexfoliation syndrome and pseudoexfoliation glaucoma and to evaluate its association with open-angle glaucoma in patients attending the Department of Ophthalmology, Dr Tomislav Bardek General Hospital in Koprivnica, northwestern Croatia. This prospective study was conducted at Dr Tomislav Bardek General Hospital between December 2012 and October 2013. A total of 5349 subjects aged 40 or above presenting for general ophthalmic examination were screened for pseudoexfoliation syndrome and pseudoexfoliation glaucoma. Each patient underwent complete ophthalmologic examination including ocular history, visual acuity testing, slit-lamp examination, applanation tonometry, optic disc evaluation, visual field analysis, and gonioscopy if glaucoma was suspected. Exclusion criteria were pseudophakic and/or aphakic patients of any age, patients with concomitant congenital eye disease, and patients with very dense ocular media opacities. Out of 5349 patients examined, there were 1994 (38.38%) males and 3201 (61.61%) females. The prevalence of pseudoexfoliation syndrome was 3.6% and primary open angle glaucoma 9.4%, out of which 23.6% with pseudoexfoliation glaucoma. The findings of this study improve our knowledge of pseudoexfoliation syndrome and pseudoexfoliation glaucoma in Croatia, particularly in the northwest region.Svrha ovoga istraživanja bila je procijeniti učestalost pseudoeksfolijativnog sindroma i pseudoeksfolijativnog glaukoma te njihovu povezanost s glaukomom otvorenog kuta kod bolesnika Očne poliklinike Opće bolnice “Dr. Tomislav Bardek” u Koprivnici, sjeverozapadna Hrvatska. Ovo prospektivno istraživanje provedeno je od prosinca 2012. do listopada 2013. godine. Ukupno je pregledano 5349 ispitanika u dobi od 40 godina i više. Kod svakog je učinjen kompletan oftalmološki pregled uključujući anamnezu, ispitivanje vidne oštrine, pregled na biomikroskopu, aplanacijsku tonometriju, pregled očnoga živca i pozadine oka te vidno polje i gonioskopiju kod onih sa sumnjom na glaukom. Kriteriji za isključenje bili su bolesnici s pseudofakijom/afakijom, bolesnici s prirođenim očnim bolestima te oni s vrlo gustim zamućenjem očnih medija. Od 5349 bolesnika bilo je 1994 (38,38%) muškaraca i 3201 (61,61%) žena. Učestalost pseudoeksfolijativnog sindroma bila je 3,6%, a primarnog glaukoma otvorenog kuta 9,4%, od čega 23,6% spada u skupinu pseudoeksfolijativnog glaukoma. Rezultati ove studije proširuju naše spoznaje o pseudoeksfolijativnom sindromu i pseudoeksfolijativnom glaukomu u Hrvatskoj, osobito u sjeverozapadnoj regiji

Using whole exome sequencing to explore genetic basis of unicuspid aortic valve disease

Normal aortic valve consists of three cusps that develop in the embryonic stage. Unicuspid aortic valve (UAV) is a rare congenital anomaly resulting in only one cusp with estimated prevalence of 0.02% in general population. Aim of this study was to identify genetic variants possibly associated with development of UAV. The study included 17 subjects, namely 5 UAV patients and their healthy family members without UAV disorder. Total DNA was isolated from venous blood samples and whole exomes sequencing (WES) was performed using BGI’s WES protocol. Adapter-trimmed and quality-filtered reads (fastp) were mapped to hg38 reference genome using BWA/SAMtools. VCF files were generated using GATK (BaseRecalibrator, HaplotypeCaller) and annotated with InterVar and AnnoVar tools. Rare heterozygous variants present in UAV patients were found in NOTCH1, TGFB2, MYH6, EGFR, FBN2, C1R, ROBO4 and TBX5, genes associated with development of aortic valves. Among these, most were missense mutations with damaging effects as predicted using in silico tools (SIFT and/or Polyphen). Only mutation in MYH6 p.Ala1130Ser was found in at least two different UAV patients. Also, rare homozygous missense mutation p.Gly577Ser with high damaging potential was found in ADAMTS5 gene. Besides, highly damaging heterozygous missense mutations were detected in gene interacting functional partners (STRING) of genes associated with development of aortic valves: DVL1, THBS1, NOTCH4, ADAMTS3, FBN1, NOTCH2, ADAM17, LRP5, WWTR1, C1S, ANKRD6 and TNNI1, as well as homozygous in ACAN and KNG1. Taken together, malfunctions in ADAMTS5, ACTA2, MYH6, FBN2, AXIN1, CELSR1 or TBX5 networks were found to be common in at least two UAV patients, suggesting existence of genetic basis in UAV disorder, possibly as a result of combined effects of multiple variants.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

A 3D in vitro cell culture model based on perfused bone-like scaffolds for healthy and pathological bone research

Comprehensive research, particularly in evaluating drug efficacy, still heavily relies on the results obtained by the utilization of cell monolayers and animals. However, the inherent limitations of these models such as their physiological disparities from humans pose significant obstacles to acquiring reliable results thus impeding further scientific progression. To address this challenge, 3D in vitro cell culture models emerged as physiologically relevant models having the potential to enhance research and drug discovery. Our study aimed to develop a 3D in vitro cell culture model based on bone-like scaffolds in conjunction with a perfusion bioreactor (“3D Perfuse”, Innovation Center FTM, Belgrade, Serbia) for studying both physiological and pathological (i.e. tumors) bone conditions

HOW NOVEL BIOMATERIALS BASED ON BIOACTIVE GLASS AND β-TRICALCIUM PHOSPHATE CAN BE EVALUATED UNDER PHYSIOLOGICALLY RELEVANT CONDITIONS?

Comprehensive preclinical studies are essential for the development of novel biomaterials that can be used in biomedical applications. However, traditional methods used for the evaluation of biomaterials have certain limitations. In vitro testing in cell monolayers is fast and easily accessible, but the 2D environment can affect cell metabolism and morphology, leading to unreliable results. On the other hand, in vivo animal studies are complex, time-consuming, expensive, and raise ethical concerns. Biomimetic bioreactors, primarily developed for tissue engineering to provide a physiologically relevant, strictly controlled environment that mimics the conditions in specific tissues or organs, could be indispensable tools in physiologically relevant biomaterial characterization and step between in vitro and in vivo studies. They offer the majority or all the necessary biochemical (e.g. pH, nutrients, gases, growth factors) and biophysical signals (e.g., shear stress, hydrostatic pressure, mechanical strains) highly relevant for biomaterial assessment and prediction of material behavior after implantation. Our group has developed two types of potential biomaterials aimed for bone and osteochondral tissue engineering based on bioactive glass (BAG), β-tricalcium phosphate (β-TCP), and different natural polymers (gellan gum and alginate). Scaffolds' integrity and mechanical properties were monitored continuously under the physiological level of mechanical compression using a dynamic compression bioreactor coupled with medium perfusion during 14 days. Formation of hydroxyapatite (HAp) within the scaffolds was investigated in a perfusion bioreactor, in the presence of simulated body fluid (SBF) during 14 and 28 days for scaffolds based on BAG and β-TCP, respectively. SEM, EDS, and XRD results have shown a significant increase in the formation of HAp under bioreactor conditions compared to static control conditions. Beyond that, formed HAp crystals were more uniformly distributed throughout scaffolds and presented more cauliflower-like morphology. The obtained results demonstrated the utilization potential of biomimetic bioreactors in physiologically relevant biomaterial characterization

Early screening for autistic spectrum disorders in toddlers aged 16 to 30 months

Introduction: Prevalence of autism spectrum disorders (ASD) worldwide shows a significant rise. Screening and early diagnosis are extremely important and provide a possibility for an early intervention. Objectives: This study is a part of validation process for a Serbian translation of Modified Checklist for Autism in Toddlers, Revised with Follow-up (MCHAT- R/F). Another aim was to analyse the risk factors which are associated with the development of ASD. Methods: The study included parents of toddlers aged 16 to 30 months during a well-child care visits in Dom zdravlja Savski venac and parents of toddlers sent to see a psychiatrist in Institute for mental health (IMH). Children were screened using the M-CHATR/ F. Results: The study included 65 toddlers. The ones screened in Dom zdravlja were placed in a low-risk group, and others who were referred to IMH were classified as high-risk group. Comparing these two groups no statistically significant difference was found in terms of children's age or parents' age at birth. Statistically significant difference was found in terms of gestational week at birth, and significantly higher incidence of pregnancy complications in high-risk group. Conclusion: Further investigation should completely examine the psychometric characteristics of M-CHAT, but the preliminary findings show that M-CHAT can detect many cases of ASD during a routine pediatrician examination. Some prenatal and perinatal factors such as pregnancy complications and gestational age at birth can lead to increased risk of developing ASD