Two rare autosomal recessive neurological disorders identified by combined genetic approaches in a single consanguineous family with multiple offspring.

INTRODUCTION: Neurodevelopmental disorders (NDDs) refer to a broad range of diseases including developmental delay, intellectual disability, epilepsy, autism spectrum disorders, and attention-deficit/hyperactivity disorder caused by dysfunctions in tightly controlled brain development. The genetic backgrounds of NDDs are quite heterogeneous; to date, recessive or dominant variations in numerous genes have been implicated. Herein, we present a large consanguineous family from Turkiye, who has been suffering from NDDs with two distinct clinical presentations. METHODS AND RESULTS: Combined in-depth genetic approaches led us to identify a homozygous frameshift variant in NALCN related to NDD and expansion of dodecamer repeat in CSTB related to Unverricht-Lundborg disease (ULD). Additionally, we sought to functionally analyze the NALCN variant in terms of mRNA expression level and current alteration. We have both detected a decrease in the level of premature stop codon-bearing mRNA possibly through nonsense-mediated mRNA decay mechanism and also an increased current in patch-clamp recordings for the expressed truncated protein. CONCLUSION: In conclusion, increased consanguinity may lead to the revealing of distinct rare neurogenetic diseases in a single family. Exome sequencing is generally considered the first-tier diagnostic test in individuals with NDD. Yet we underline the fact that customized approaches other than exome sequencing may be used as in the case of ULD to aid diagnosis and better genetic counseling.

Perception and management of cancer predisposition in pediatric cancer centers: A European-wide questionnaire-based survey.

The European Union-funded COST Action (LEukaemia GENe Discovery by data sharing, mining, and collaboration) LEGEND was an international and multidisciplinary collaboration between clinicians and researchers that covered a range of aspects of genetic predisposition in childhood leukemia. Within this framework, we explored the perception and handling of genetic predisposition in the daily practice of European treatment centers. Herein, we present the results of our questionnaire-based survey. We found that the overall awareness is quite high, and respondents remarked that identification and treatment of the most common predisposition syndromes were present. Nevertheless, high demand for continuous education and routinely updated resources remains.

Impact of TP53 gene variants on prognosis and survival of childhood acute lymphoblastic leukemia.

The tumor suppressor protein 53 (TP53) gene is one of the most studied genes in cancer. Although TP53 variants are rare events in acute leukemia, recent observations showed that relapse samples might harbor TP53 variants. Here, we aimed to determine TP53 variants (hotspot region, exon 4-11) in childhood acute lymphoblastic leukemia (B and T-ALL) patients (n = 94) including diagnostic-relapse pairs (n = 15) by amplicon sequencing and evaluate the clinical impact of these variants. In total, nine different (E298*, R283C, R273H, L252F, C229F, I195T, E286G, c.955_956insC, and c.920-1G > C) variants were identified in 17 (18%) childhood ALL patients. c.(920-1G> C) splice site variant and c.(955_956insC) insertion were reported for the first time. In diagnose-relapse pair samples, we identified acquired and/or loss of TP53 variants in the samples at the time of relapse. TP53 variants were found to be more common in T-ALL (37%) than in B-ALL patients (9%). Pathogenic TP53 variants were associated with a shorter overall survival time (p = 0.001).TP53 variants were found to be associated with inferior outcomes in our cohort and can be an independent risk factor for poor prognosis in childhood acute leukemia patients. Identification of low-frequent variants with next-generation sequencing approaches enables better knowledge of the clonal dynamics of ALL.

Digital distraction levels of university students in emergency remote teaching.

The main objective of this study is to investigate the relationships between digital distraction, perceived learning, and general satisfaction in emergency remote teaching. Correlational design, one of the quantitative research methods, was used for the study. The study sample consists of 1532 university students in a Turkish university during Covid-19. The study results show a significant relationship between digital distraction, general satisfaction, and perceived learning. Digital distraction is negatively related to general satisfaction and perceived learning, and general satisfaction is positively related to perceived learning. When the independent demographic variables were analyzed, digital distraction scores were higher for females, those not working in any job, not participate orientation training, and not following the live class and watching it later. In addition, it was revealed that as the age of the students decreased, the digital distraction scores increased. It was found that digital distraction variables, the amount of digital distraction, sending instant messages, checking the time, boredom, sharing social media, and system usability were significant predictors of digital distraction.

Determining T and B Cell development by TREC/KREC analysis in primary immunodeficiency patients and healthy controls.

T cell receptor excision circles (TRECs) and kappa-deleting excision circles (KRECs) are DNA fragments potentially indicative of T and B cell development, respectively. Recent thymic emigrants (RTEs) are a subset of peripheral cells that may also represent thymic function. Here, we investigated TREC/KREC copy numbers by quantitative real-time PCR in the peripheral blood of patients with primary immunodeficiencies (PIDs, n = 145) and that of healthy controls (HCs, n = 86) and assessed the correlation between RTEs and TREC copy numbers. We found that TREC copy numbers were significantly lower in children and adults with PIDs (P < .0001 and P < .002, respectively) as compared with their respective age-matched HCs. A moderate correlation was observed between TREC copies and RTE numbers among children with PID (r = .5114, P < .01), whereas no significant correlation was detected between RTE values and TREC content in the HCs (r = .0205, P = .9208). Additionally, we determined TREC and KREC copy numbers in DNA isolated from the Guthrie cards of 200 newborns and showed that this method is applicable to DNA isolated from both peripheral blood samples and dried blood spots, with the two sample types showing comparable TREC and KREC values. We further showed that RTE values are not always reliable markers of T cell output. Although additional confirmatory studies with larger cohorts are needed, our results provide thresholds for TREC/KREC copy numbers for different age groups.

Should we take precautions to avoid respiratory compromise while delaying CPAP resumption following transsfenoidal surgery? An alternative approach in a patient with severe obstructive sleep apnea: case report.

BACKGROUND: We describe a patient with severe obstructive sleep apnea scheduled for transsfenoidal surgery. Early postoperative use of continuous positive airway pressure (CPAP) was considered unsafe because increased risk of intracranial complications. METHODS: Aiming to bypass the upper airway obstruction and thus avoid CPAP, a 6-mm nasopharyngeal airway was introduced by the surgical team under endoscopic vision. In the postoperative period and during follow-up, patient and his family did not complain about apnea/hypopnea episodes and nasopharyngeal airway was tolerated comfortably. CONCLUSION: We recommend this technique as an alternative in obstructive sleep apnea patients undergoing transsfenoidal surgery.

The rare rs769301934 variant in NHLRC1 is a common cause of Lafora disease in Turkey.

Lafora disease (LD) is a severe form of progressive myoclonus epilepsy inherited in an autosomal recessive fashion. It is associated with biallelic pathogenic variations in EPM2A or NHLRC1, which encode laforin and malin, respectively. The disease usually starts with adolescent onset seizures followed by progressive dementia, refractory status epilepticus and eventually death within 10 years of onset. LD is generally accepted as having a homogenous clinical course with no considerable differences between EPM2A or NHLRC1 associated forms. Nevertheless, late-onset and slow progressing forms of the disease have also been reported. Herein, we have performed clinical and genetic analyses of 14 LD patients from 12 different families and identified 8 distinct biallelic variations in these patients. Five of these variations were novel and/or associated with the LD phenotype for the first time. Interestingly, almost half of the cases were homozygous for the rare rs769301934 (NM_198586.3(NHLRC1): c.436 G > A; p.(Asp146Asn)) allele in NHLRC1. A less severe phenotype with an onset at a later age may be the reason for the biased inflation of this variant, which is already present in the human gene pool and can hence arise in the homozygous form in populations with increased parental consanguinity.

Mutational landscape of severe combined immunodeficiency patients from Turkey.

Severe combined immunodeficiency (SCID) has a diverse genetic aetiology, where a clinical phenotype, caused by single and/or multiple gene variants, can give rise to multiple presentations. The advent of next-generation sequencing (NGS) has recently enabled rapid identification of the molecular aetiology of SCID, which is crucial for prognosis and treatment strategies. We sought to identify the genetic aetiology of various phenotypes of SCIDs and assessed both clinical and immunologic characteristics associated with gene variants. An amplicon-based targeted NGS panel, which contained 18 most common SCID-related genes, was contumely made to screen the patients (n = 38) with typical SCID, atypical SCID or OMENN syndrome. Allelic segregations were confirmed for the detected gene variants within the families. In total, 24 disease-causing variants (17 known and 7 novel) were identified in 23 patients in 9 different SCID genes: RAG1 (n = 5), RAG2 (n = 2), ADA (n = 3), DCLRE1C (n = 2), NHEJ1 (n = 2), CD3E (n = 2), IL2RG (n = 3), JAK3 (n = 4) and IL7R (n = 1). The overall success rate of our custom-made NGS panel was 60% (39.3% for NK+ SCID and 100% for NK- SCID). Incidence of autosomal-recessive inherited genes is more frequently found in our cohort than the previously reported populations probably due to the high consanguineous marriages in Turkey. In conclusion, the custom-made sequencing panel was able to identify and confirm the previously known and novel disease-causing variants with high accuracy.

Biallelic loss of EEF1D function links heat shock response pathway to autosomal recessive intellectual disability.

Intellectual disability (ID) is a genetically heterogeneous neurodevelopmental disorder characterised by significantly impaired intellectual and adaptive functioning. ID is commonly syndromic and associated with developmental, metabolic and/or neurological findings. Autosomal recessive ID (ARID) is a significant component of ID especially in the presence of parental consanguinity. Several ultra rare ARID associated variants in numerous genes specific almost to single families have been identified by unbiased next generation sequencing technologies. However, most of these new candidate ARID genes have not been replicated in new families due to the rarity of associated alleles in this highly heterogeneous condition. To determine the genetic component of ARID in a consanguineous family from Turkey, we have performed SNP-based linkage analysis in the family along with whole exome sequencing (WES) in an affected sibling. Eventually, we have identified a novel pathogenic variant in EEF1D, which has recently been recognised as a novel candidate gene for ARID in a single family. EEF1D encodes a ubiquitously expressed translational elongation factor functioning in the cytoplasm. Herein, we suggest that the loss of function variants exclusively targeting the long EEF1D isoform may explicate the ARID phenotype through the heat shock response pathway, rather than interfering with the canonical translational elongation.

No evidence for a BRD2 promoter hypermethylation in blood leukocytes of Europeans with juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is a common syndrome of genetic generalized epilepsies (GGEs). Linkage and association studies suggest that the gene encoding the bromodomain-containing protein 2 (BRD2) may increase risk of JME. The present methylation and association study followed up a recent report highlighting that the BRD2 promoter CpG island (CpG76) is differentially hypermethylated in lymphoblastoid cells from Caucasian patients with JME compared to patients with other GGE subtypes and unaffected relatives. In contrast, we found a uniform low average percentage of methylation (<4.5%) for 13 CpG76-CpGs in whole blood cells from 782 unrelated European Caucasians, including 116 JME patients, 196 patients with genetic absence epilepsies, and 470 control subjects. We also failed to confirm an allelic association of the BRD2 promoter single nucleotide polymorphism (SNP) rs3918149 with JME (Armitage trend test, P = 0.98), and we did not detect a substantial impact of SNP rs3918149 on CpG76 methylation in either 116 JME patients (methylation quantitative trait loci [meQTL], P = 0.29) or 470 German control subjects (meQTL, P = 0.55). Our results do not support the previous observation that a high DNA methylation level of the BRD2 promoter CpG76 island is a prevalent epigenetic motif associated with JME in Caucasians.

Homozygous c.130-131 ins A (pW44X) mutation in the HAX1 gene as the most common cause of congenital neutropenia in Turkey: Report from the Turkish Severe Congenital Neutropenia Registry.

BACKGROUND: Severe congenital neutropenia is a rare disease, and autosomal dominantly inherited ELANE mutation is the most frequently observed genetic defect in the registries from North America and Western Europe. However, in eastern countries where consanguineous marriages are common, autosomal recessive forms might be more frequent. METHOD: Two hundred and sixteen patients with severe congenital neutropenia from 28 different pediatric centers in Turkey were registered. RESULTS: The most frequently observed mutation was HAX1 mutation (n = 78, 36.1%). A heterozygous ELANE mutation was detected in 29 patients (13.4%) in our cohort. Biallelic mutations of G6PC3 (n = 9, 4.3%), CSF3R (n = 6, 2.9%), and JAGN1 (n = 2, 1%) were also observed. Granulocyte colony-stimulating factor treatment was given to 174 patients (80.6%). Two patients died with infectious complications, and five patients developed myelodysplastic syndrome/acute myeloblastic leukemia. The mean (± mean standard error) follow-up period was 129.7 ± 76.3 months, and overall survival was 96.8% (CI, 94.4-99.1%) at the age of 15 years. In Turkey, severe congenital neutropenia mostly resulted from the p W44X mutation in the HAX1 gene. CONCLUSION: In Turkey, mutation analysis should be started with HAX1, and if this is negative, ELANE and G6PC3 should be checked. Because of the very high percentage of consanguineous marriage, rare mutations should be tested in patients with a negative mutation screen.

Clinical and genetic spectrum of an orphan disease MPAN: a series with new variants and a novel phenotype.

INTRODUCTION: Pathogenic variations in C19orf12 are responsible for two allelic diseases: mitochondrial membrane protein-associated neurodegeneration (MPAN); and spastic paraplegia type 43 (SPG43). MPAN is an orphan disease, which presents with spasticity, dystonia, peripheral nerve involvement, and dementia. The pattern of iron accumulation on brain MRI may be a clue for the diagnosis of MPAN. SPG43, on the other hand, is characterised by progressive lower limb spasticity without brain iron accumulation. We here present clinical and genetic findings of MPAN patients with potentially pathogenic C19orf12 variants. MATERIALS AND METHODS: Patients from 13 different families having progressive motor symptoms with irritative pyramidal signs and brain iron accumulation were screened for C19orf12 gene variants. RESULTS: C19orf12 screening identified seven variants associated with MPAN in eight patients from seven families. We associated two pathogenic variants (c.24G > C; p.(Lys8Asn) and c.194G > A; p.(Gly65Glu)) with the MPAN phenotype for the first time. We also provided a genetic diagnosis for a patient with an atypical MPAN presentation. The variant c.32C > T; p.(Thr11Met), common to Turkish adult-onset MPAN patients, was also detected in two unrelated late-onset MPAN patients. CONCLUSIONS: Genetic analysis along with thorough clinical analysis supported by radiological findings will aid the differential diagnosis of MPAN within the neurodegeneration with brain iron accumulation spectrum as well as other disorders including hereditary spastic paraplegia. Dystonia and parkinsonism may not be the leading clinical findings in MPAN patients, as these are absent in the atypical case. Finally, we emphasise that the existence of frameshifting variants may bias the age of onset toward childhood.

A novel pathogenic frameshift variant of CD3E gene in two T-B+ NK+ SCID patients from Turkey.

Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency, which is characterized by the dysfunction and/or absence of T lymphocytes. Early diagnosis of SCID is crucial for overall survival, and if it remains untreated, SCID is often fatal. Next-generation sequencing (NGS) has become a rapid, high-throughput technology, and has already been proven to be beneficial in medical diagnostics. In this study, a targeted NGS panel was developed to identify the genetic variations of SCID by using SmartChip-TE technology, and a novel pathogenic frameshift variant was found in the CD3E gene. Sanger sequencing has confirmed the segregation of the variant among patients. We found a novel deletion in the CD3E gene (NM000733.3:p.L58Hfs*9) in two T-B+ NK+ patients. The variant was not found in the databases of dbSNP, ExAC, and 1000G. One sibling in family I was homozygous and the rest of the family members were heterozygous for this variant. T cell receptor excision circle (TREC) and kappa-deleting recombination excision circle (KREC) analyses were performed for T and B cell maturation. TRECs were not detected in both patients and the KREC copy numbers were similar to the other family members. In addition, heterozygous family members showed decreased TREC levels when compared with the wild-type sibling, indicating that carrying this variant in one allele does not cause immunodeficiency, but does effect T cell proliferation. Here, we report a novel pathogenic frameshift variant in CD3E gene by using targeted NGS panel.

The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas.

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

herg1b expression as a potential specific marker in pediatric acute myeloid leukemia patients with HERG 897K/K genotype.

Human ether-a-go-go related gene (herg) encoding HERG K(+) channel has been demonstrated in many previous studies with its association to cell cycle progression and growth in tumor cells. The upregulated expression of HERG K+ channels was determined in different tumor types. Furthermore, not only full-length transcript herg1 but also a truncated isoform herg1b was shown to be expressed in cancer cells. In this study, the expression levels of herg1 and herg1b and the impact of K897T mutation on their expressions were investigated in pediatric acute myeloid leukemia (pAML). Expression levels of herg1 and herg1b isoforms were analyzed by quantitative real time polymerase chain reaction (PCR) in pAML patients together with healthy donors, and their expressions were confirmed by western blotting. The 2690 A>C nucleotide variation in KCNH2 gene corresponding to K897T amino acid change was analyzed by PCR followed by restriction enzyme digestion. herg1b overexpression was observed in tumor cells compared to healthy controls (P = .0024). However, herg1 expression was higher in healthy control cells than tumor cells (P = .001). The prevalence of polymorphic allele 897T was 26% in our patient group and 897T carriers showed increased herg1b expression compared to wild-type allele carriers. Our results demonstrate the presence of the increased levels of herg1b expression in pAML. In addition, we report for the first time that, pAML subgroup with HERG 897K/K genotype compared to 897K/T and T/T genotypes express increased levels of herg1b. In conclusion, HERG 897K/K genotype with increased level of herg1b expression might well be a prognostic marker for pAML.

Local Renin-Angiotensin system in normal hematopoietic and multiple myeloma-related progenitor cells.

OBJECTIVE: The prominent functions of the local renin-angiotensin system (RAS) in primitive hematopoiesis further support the hypothesis that local autocrine bone marrow RAS could also be active in neoplastic hematopoiesis. The aim of this study is to examine critical RAS elements in normal CD34+ hematopoietic stem cells and multiple myeloma (MM)-related progenitor cells. MATERIALS AND METHODS: The study group comprised the total bone marrow cells (CBM) of 10 hematologically normal people, the CD34+ stem cell samples (CD34+CBM) of 9 healthy donors for allogeneic peripheral stem cell transplantation, and the CD34+ stem cell samples (CD34+MM) of 9 MM patients undergoing autologous peripheral stem cell transplantation. We searched for the gene expression of the major RAS components in healthy hematopoietic cells and myeloma cells by quantitative real-time polymerase chain reaction analysis. RESULTS: RENIN, angiotensinogen (ANGTS), and angiotensin converting enzyme-I (ACE I) mRNA expression levels of CBM were significantly higher than those in myeloma patients (p=0.03, p=0.002, and p=0.0008, respectively). Moreover, RENIN and ANGTS mRNA expression levels were significantly higher in CD34+ stem cell samples of healthy allogeneic donors compared to those in myeloma patients (p=0.001 and p=0.01). However, ACE I expression levels were similar in CD34+CBM and CD34+MM hematopoietic cells (p=0.89). CONCLUSION: Although found to be lower than in the CBM and CD34+CBM hematopoietic cells, the local RAS components were also expressed in CD34+MM hematopoietic cells. This point should be kept in mind while focusing on the immunobiology of MM and the processing of autologous cells during the formation of transplantation treatment protocols.

Memantine and SKF82958 but not an enriched environment modulate naloxone-precipitated morphine abstinence syndrome without affecting hippocampal tPA mRNA levels in rats.

There is accumulating evidence supporting the involvement of tissue-plasminogen activator (tPA) in the mechanisms underlying the effects of morphine and an enriched environment. This study was designed to investigate possible interactive roles of the glutamatergic and the dopaminergic systems regarding hippocampal tPA in the neurobiology of morphine dependence. For this purpose, Wistar albino rats, housed in either a standard- (SE) or an enriched environment (EE) were implanted subcutaneously with morphine (150 mg base) or placebo pellets. Behavioral and somatic signs of morphine abstinence precipitated by an opioid-receptor antagonist naloxone (1 mg/kg, i.p.) 72 h after the pellet implantation were observed individually for 15 min in all groups. Memantine (10 mg/kg i.p.), an antagonist of N-methyl-D-aspartic acid class of glutamatergic receptor-subtype decreased teeth-chattering, ptosis, diarrhea and the loss of body weight. SKF82958 (1 mg/kg, i.p.), a dopamine D1-receptor agonist decreased jumping and ptosis but increased rearing and loss of body weight. On the other hand, co-administration of SKF82958 with memantine prevented some of their effects that occur when administered alone at the same doses. Furthermore, the EE did not change the intensity of morphine abstinence. The level of hippocampal tPA mRNA was found to be lower in the SE morphine abstinence group than in the placebo group and close to the EE morphine abstinence group, whereas there was no significant alteration of its level in the memantine or SKF82958 groups. These findings suggest that the interaction between the glutamatergic and the dopaminergic systems may be an important component of the neurobiology of morphine dependence, and the role of tPA in this interaction should be further investigated.

Cosmic Whirl: Navigating the Comet Trail in DNA: H2AX Phosphorylation and the Enigma of Uncertain Significance Variants.

Pathogenic variations in the BRCA2 gene have been detected with the development of next-generation sequencing (NGS)-based hereditary cancer panel testing technology. It also reveals an increasing number of variants of uncertain significance (VUSs). Well-established functional tests are crucial to accurately reclassifying VUSs for effective diagnosis and treatment. We retrospectively analyzed the multi-gene cancer panel results of 922 individuals and performed in silico analysis following ClinVar classification. Then, we selected five breast cancer-diagnosed patients' missense BRCA2 VUSs (T1011R, T1104P/M1168K, R2027K, G2044A, and D2819) for reclassification. The effects of VUSs on BRCA2 function were analyzed using comet and H2AX phosphorylation (γH2AX) assays before and after the treatment of peripheral blood mononuclear cells (PBMCs) of subjects with the double-strand break (DSB) agent doxorubicin (Dox). Before and after Dox-induction, the amount of DNA in the comet tails was similar in VUS carriers; however, notable variations in γH2AX were observed, and according to combined computational and functional analyses, we reclassified T1001R as VUS-intermediate, T1104P/M1168K and D2819V as VUS (+), and R2027K and G2044A as likely benign. These findings highlight the importance of the variability of VUSs in response to DNA damage before and after Dox-induction and suggest that further investigation is needed to understand the underlying mechanisms.

Betulin Stimulates Osteogenic Differentiation of Human Osteoblasts-Loaded Alginate-Gelatin Microbeads.

Osteoporosis, a terminal illness, has emerged as a global public health problem in recent years. The long-term use of bone anabolic drugs to treat osteoporosis causes multi-morbidity in elderly patients. Alternative therapies, such as allogenic and autogenic tissue grafts, face important issues, such as a limited source of allogenic grafts and tissue rejection in autogenic grafts. However, stem cell therapy has been shown to increase bone regeneration and decrease osteoporotic bone formation. Stem cell therapy combined with betulin (BET) supplementation might be adequate for bone remodeling and new bone tissue generation. In this study, the effect of BET on the viability and osteogenic differentiation of hFOB 1.19 cells was investigated. The cells were encapsulated in alginate-gelatin (AlGel) microbeads. In vitro tests were conducted during the 12 d of incubation. While BET showed cytotoxic activity (>1 µM) toward non-encapsulated hFOB 1.19 cells, encapsulated cells retained their functionality for up to 12 days, even at 5 µM BET. Moreover, the expression of osteogenic markers indicates an enhanced osteo-inductive effect of betulin on encapsulated hFOB 1.19, compared to the non-encapsulated cell culture. The 3D micro-environment of the AlGel microcapsules successfully protects the hFOB 1.19 cells against BET cytotoxicity, allowing BET to improve the mineralization and differentiation of osteoblast cells.

Preparation of Cell-Loaded Microbeads as Stable and Injectable Delivery Platforms for Tissue Engineering.

Cell transplants in therapeutic studies do not preserve their long-term function inside the donor body. In mesenchymal stem cell (MSC) transplants, transplanted cells disperse through the body and are prone to degradation by immune cells after the transplant process. Various strategies, such as usage of the immunosuppressive drugs to eliminate allograft rejection, are designed to increase the efficiency of cell therapy. Another strategy is the construction of biomimetic encapsulates using polymeric materials, which isolate stem cells and protect them from environmental effects. In this study, fibroblasts (L929) and MSCs were investigated for their improved viability and functionality once encapsulated inside the alginate microbeads under in vitro conditions for up to 12 days of incubation. Thus, uniform and injectable (<200 µm) cell-loaded microbeads were constructed by the electrostatically assisted spraying technique. Results showed that both L929 and MSCs cells continue their metabolic activity inside the microbeads during the incubation periods. Glucose consumption and lactic acid production levels of both cell lines were consistently observed. The released cell number on day 12 was found to be increased compared to day 0. Protein expression levels of both groups increased every day with the expected doubling rate. Hence, this strategy with a simple yet clever design to encapsulate either MSCs or L929 cells might outstand as a potential cell delivery platform for cell therapy-based tissue engineering.