OView-AI Supporter for Classifying Pneumonia, Pneumothorax, Tuberculosis, Lung Cancer Chest X-ray Images Using Multi-Stage Superpixels Classification.

The deep learning approach has recently attracted much attention for its outstanding performance to assist in clinical diagnostic tasks, notably in computer-aided solutions. Computer-aided solutions are being developed using chest radiography to identify lung diseases. A chest X-ray image is one of the most often utilized diagnostic imaging modalities in computer-aided solutions since it produces non-invasive standard-of-care data. However, the accurate identification of a specific illness in chest X-ray images still poses a challenge due to their high inter-class similarities and low intra-class variant abnormalities, especially given the complex nature of radiographs and the complex anatomy of the chest. In this paper, we proposed a deep-learning-based solution to classify four lung diseases (pneumonia, pneumothorax, tuberculosis, and lung cancer) and healthy lungs using chest X-ray images. In order to achieve a high performance, the EfficientNet B7 model with the pre-trained weights of ImageNet trained by Noisy Student was used as a backbone model, followed by our proposed fine-tuned layers and hyperparameters. Our study achieved an average test accuracy of 97.42%, sensitivity of 95.93%, and specificity of 99.05%. Additionally, our findings were utilized as diagnostic supporting software in OView-AI system (computer-aided application). We conducted 910 clinical trials and achieved an AUC confidence interval (95% CI) of the diagnostic results in the OView-AI system of 97.01%, sensitivity of 95.68%, and specificity of 99.34%.

Deep Learning in Multi-Class Lung Diseases' Classification on Chest X-ray Images.

Chest X-ray radiographic (CXR) imagery enables earlier and easier lung disease diagnosis. Therefore, in this paper, we propose a deep learning method using a transfer learning technique to classify lung diseases on CXR images to improve the efficiency and accuracy of computer-aided diagnostic systems' (CADs') diagnostic performance. Our proposed method is a one-step, end-to-end learning, which means that raw CXR images are directly inputted into a deep learning model (EfficientNet v2-M) to extract their meaningful features in identifying disease categories. We experimented using our proposed method on three classes of normal, pneumonia, and pneumothorax of the U.S. National Institutes of Health (NIH) data set, and achieved validation performances of loss = 0.6933, accuracy = 82.15%, sensitivity = 81.40%, and specificity = 91.65%. We also experimented on the Cheonan Soonchunhyang University Hospital (SCH) data set on four classes of normal, pneumonia, pneumothorax, and tuberculosis, and achieved validation performances of loss = 0.7658, accuracy = 82.20%, sensitivity = 81.40%, and specificity = 94.48%; testing accuracy of normal, pneumonia, pneumothorax, and tuberculosis classes was 63.60%, 82.30%, 82.80%, and 89.90%, respectively.

Clinical characteristics and associated factors of canal switch in benign paroxysmal positional vertigo.

BACKGROUND: Canal switch benign paroxysmal positional vertigo (CS-BPPV) is a transition of BPPV involving one canal to another canal during or after canalith repositioning procedures (CRP). OBJECTIVE: To investigate the clinical characteristics of CS-BPPV and its associated factors. METHODS: The data of 2,303 patients with BPPV involving the lateral canal (LC) or posterior canal (PC) were retrospectively analyzed. Demographics, etiologies, and various clinical parameters related to CRP were compared between patients with and without CS-BPPV. RESULTS: Sixty-eight (2.95%) patients exhibited CS-BPPV. For patients with CS-BPPV from the PC to the LC, as well as those with CS-BPPV from the LC to the PC, the CRP number for the original canal in CS-BPPV was significantly greater than in non-CS-BPPV (P = 0.002). More CRP cycles were required to treat CS-BPPV than non-CS-BPPV involving the same canal. Multivariate analysis showed that CS-BPPV from the LC to the PC was significantly associated with multiple CRP cycles and use of the Gufoni maneuver (P = 0.038 and P < 0.001, respectively). CONCLUSIONS: The use of multiple cycles of CRP and the Gufoni maneuver were significantly associated with the onset of CS-BPPV. Furthermore, more CRP cycles were needed for the treatment of CS-BPPV than for non-CS-BPPV involving the same canal.

Patterns of nystagmus conversion in sudden sensorineural hearing loss with vertigo.

The patients with sudden sensorineural hearing loss (SSNHL) may complain of vertigo. Although there have been many reports on SSNHL with vertigo (SSNHL_V), changes in the pattern of nystagmus have not been studied as yet. This study is a retrospective study and aims to investigate the characteristic changes in type of nystagmus and clinical features in patients with SSNHL_V who experienced a change in their nystagmus pattern during follow-up. Among 50 patients with SSNHL_V between January 2012 and December 2015, we identified 15 patients with SSNHL_V whose pattern of nystagmus changed. Initial nystagmus was classified into 5 subgroups: paretic type, irritative type, persistent geotropic direction-changing positional nystagmus (PG-DCPN), persistent apogeotropic direction-changing positional nystagmus (PA-DCPN), and posterior semicircular canal benign paroxysmal positional vertigo. The most common pattern of initial nystagmus was PG-DCPN (n = 7). The change of initial nystagmus pattern occurred on day 2 to 75 from symptom onset, and 2 (of 15) patients showed further conversion. The most common pattern of final nystagmus was PA-DCPN (n = 9). Hearing improvement after treatment was not significantly different (P = .59) between SSNHL_V patients with nystagmus change (25 ±â€Š17 dB, n = 15) and those without nystagmus change (28 ±â€Š18 dB, n = 35). In conclusion, clinician's attention is required in evaluating the vertigo symptom in patients with SSNHL_V because the initial patterns of nystagmus can be converted to another type of nystagmus. The presence of nystagmus change during follow-up may not be a prognosticator for hearing recovery in patients with SSNHL_V.

Nystagmus Findings and Hearing Recovery in Idiopathic Sudden Sensorineural Hearing Loss Without Dizziness.

OBJECTIVES: To investigate the incidence of nystagmus in patients with sudden sensorineural hearing loss (SSNHL) without dizziness, and to evaluate the correlation of the presence of nystagmus with hearing recovery in those patients. STUDY DESIGN: Retrospective study. SETTING: Tertiary referral academic medical center. PATIENTS: Thirty-eight patients with SSNHL who did not complained of dizziness were enrolled. MAIN OUTCOME MEASURE: The presence of nystagmus was evaluated by recording eye movements, and if present, nystagmus was classified as direction-fixed or positional nystagmus. Vestibular function tests were performed, including caloric test. RESULTS: Of 33 idiopathic SSNHL patients without dizziness, nystagmus was observed in 22 patients (67%), of which 14 patients exhibited direction-fixed nystagmus, and 8 patients exhibited direction-changing nystagmus. Among the 14 patients with direction-fixed nystagmus, 9 displayed the paretic type, and 5 displayed the irritative type. Direction-changing nystagmus (n = 8) was defined as that in which the direction of nystagmus was changed in a supine head-roll test, and the geotropic type and apogeotropic type were observed in two and six patients, respectively. The mean initial pure tone threshold was 58.2 ±â€Š28.1 dB and 57.3 ±â€Š20.0 dB in SSNHL patients with and without nystagmus, respectively, which was not significantly different (p = 0.925). When hearing improvement was compared according to the presence of nystagmus, 39% (8 of 21) of patients with nystagmus were found to belong in the good prognosis group, and 72% (8 of 11) of patients without nystagmus were found to belong in the good prognosis group, which showed marginal statistical significance (p = 0.063). CONCLUSION: Nystagmus was observed in two-thirds of idiopathic SSNHL patients without dizziness, and the pattern of nystagmus was either direction fixed or direction changing. Because hearing recovery was worse in patients with nystagmus than those without it, the diagnosis of nystagmus, even in SSNHL patients without dizziness, may be important in evaluating the hearing prognosis.

Utility of the bow and lean test in predicting subtype of benign paroxysmal positional vertigo.

OBJECTIVES/HYPOTHESIS: To investigate the role of the bow and lean test (BLT) in the diagnosis of benign paroxysmal positional vertigo (BPPV). STUDY DESIGN: Retrospective case-control study. METHODS: Between March 2015 and June 2017, we enrolled 113 patients with posterior semicircular canal (PSCC) BPPV, 74 patients with lateral semicircular canal (LSCC) canalolithiasis, 53 patients with LSCC cupulolithiasis, and 32 patients with light cupula. We retrospectively assessed bowing nystagmus (BN) and leaning nystagmus (LN). RESULTS: In PSCC BPPV, 75% of the patients showed at least one of BN and LN, and direction of nystagmus provoked by a Dix-Hallpike test on the affected side was consistent with that of LN and opposite to that of BN. In LSCC canalolithiasis, 65% (48 of 74) of the patients showed both BN and LN, which were in the same direction in 38 patients (of 48) and in the opposite direction in 10 patients (of 48). The affected side can be determined according to the results of THE BLT in 74% (55 of 74) of LSCC canalolithiasis patients, and among them, the side determined according to the results of head-roll test was discordant with that according to the BLT in 20 of 55 patients (36%). In LSCC cupulopathy (n = 85), both BN and LN were persistent and observed in all cases, but we could not distinguish LSCC cupulolithiasis from light cupula according to nystagmus direction in the BLT. CONCLUSIONS: Although a BLT yields better lateralization in LSCC canalolithiasis, it may be more useful in predicting the diagnosis and lateralization of PSCC BPPV than LSCC canalolithiasis. LEVEL OF EVIDENCE: 4 Laryngoscope, 2600-2604, 2018.

The Light Cupula: An Emerging New Concept for Positional Vertigo.

Benign paroxysmal positional vertigo (BPPV) is the most common type of positional vertigo. A canalolithiasis-type of BPPV involving the lateral semicircular canal (LSCC) shows a characteristic direction-changing positional nystagmus (DCPN) which beats towards the lower ear (geotropic) on turning the head to either side in a supine position. Because geotropic DCPN in LSCC canalolithiasis is transient with a latency of a few seconds, the diagnosis can be challenging if geotropic DCPN is persistent without latency. The concept of "light cupula" has been introduced to explain persistent geotropic DCPN, although the mechanism behind it requires further elucidation. In this review, we describe the characteristics of the nystagmic pattern in light cupula and discuss the current evidence for possible mechanisms explaining the phenomenon.

Pseudo-spontaneous nystagmus in horizontal semicircular canal canalolithiasis.

Benign paroxysmal positional vertigo (BPPV) involving horizontal semicircular canal (HSCC) is characterized by direction-changing positional nystagmus (DCPN) in a supine roll test, and the occurrence of spontaneous nystagmus in HSCC BPPV has been reported recently. The aim of this study is to investigate the characteristics of pseudo-spontaneous nystagmus (PSN) in patients with HSCC canalolithiasis, and evaluate the effect of the presence of PSN on treatment outcome.Between April 2014 and January 2016, 75 and 59 patients with HSCC canalolithiasis and cupulolithiasis, respectively, were enrolled. Spontaneous and positional nystagmus were examined.PSN was observed in 31 of 75 patients (41%) with HSCC canalolithiasis, and 55 of 59 patients (93%) with HSCC cupulolithiasis. PSN persisted during the period of observation, which was at least 1 minute in all patients with PSN. In HSCC canalolithiasis, direction-reversing nystagmus was observed in 58 patients (25 bilateral and 33 unilateral). Nine of 25 patients with bilateral direction-reversing nystagmus, and 22 of 33 patients with unilateral direction-reversing nystagmus showed PSN. None of 17 patients without direction-reversing nystagmus showed PSN. The direction of PSN corresponded to that of direction-reversing nystagmus in all 22 patients with unilateral direction-reversing nystagmus. The proportion of patients who recovered after 1 session of repositioning maneuver was not significantly different between patients with and without PSN (P = .867).PSN was observed more commonly in HSCC cupulolithiasis than canalolithiasis. The pathophysiologic mechanism underlying PSN can be explained by natural inclination of HSCC and medial to lateral orientation of the HSCC cupular axis in cupulolithiasis, and by spontaneous reversal of initial positional nystagmus (direction-reversing nystagmus) generated by short-term adaptation of vestibulo-ocular reflex in canalolithiasis. The presence of PSN in HSCC canalolithiasis may not affect the treatment outcome.

The Effect of Sleep Disordered Breathing on Olfactory Functions: Analysis by Apnea-Hypopnea Index.

OBJECTIVES: One hypothesis of obstructive sleep apnea syndrome (OSAS) is that long-standing snoring vibrations and hypoxia of the nerves cause a local neuropathy in the upper airway during sleep. The aim of this study was to investigate olfactory function in subjects comprising snorers and untreated subjects with OSAS, and to correlate data with polysomnographic parameters. METHODS: Sixty-nine patients were evaluated for snoring from January 2010 to December 2013. The mild group (apneahypopnea index [AHI]<15) consisted of 19 subjects, and the moderate-severe group (AHI≥15) consisted of 50 subjects. Exclusion criteria were conductive olfactory dysfunction, previous tonsil or soft palatal surgery, central sleep apnea, and medications that are known to affect peripheral nerves. Nocturnal polysomnography and olfactory function test such as Korean version of Sniffin's stick test I, II (KVSS I, II) were performed. RESULTS: There was a significant difference in body mass index, average oxygen saturation (SaO2), lowest SaO2, average snoring duration, and KVSS I, II between the two groups. AHI was related to odor threshold score, and average SaO2 was related to odor discrimination score. But, odor identification score showed no relation with AHI and average SaO2 except for age. Average SaO2 and AHI were closely related to the function of smell. CONCLUSION: Hypoxia and low nasal airflow caused by OSAS may have an effect on the olfactory function. On comparison between the two groups, patients with a high AHI, especially those with OSAS, had an olfactory dysfunction. Also, low average oxygen is the main risk factor in determining the olfactory function. In people with OSAS, the possibility of olfactory dysfunction should be considered and an olfactory function test should be performed.

Tumor-Targeting Co-Delivery of Drug and Gene from Temperature-Triggered Micelles.

Co-delivery strategy using multifunctional nanocarriers is an attractive option for the synergistic and enhanced effects in cancer treatment, but one system integrating multiple functions for controlled release at the target is still challenging. Herein, this study shows the synthesis and characterization of our stimulus-responsive co-delivery system for the controlled release into tumors, which is composed of polyethylenimine (PEI)-linked Pluronic F127 (PF127) and folic acid (FA), called PF127-PEI-FA. PF127-PEI-FA system facilitated drug loading and gene complex formation, and showed controlled release behaviors in response to hitting temperature to hyperthermia. PF127-PEI-FA system was demonstrated to be biocompatible and showed receptor-mediated gene delivery. The results of our multifunctional nanocarrier system that enabled co-delivery suggest a promising potential for controlled drug release at targeted areas. However, further in-depth studies on the use of therapeutic drugs and genes in multiple cell types and the animal response are required.

Structure, biodegradation behavior and cytotoxicity of alkali-containing alkaline-earth phosphosilicate glasses.

We report on the effect of sodium on the structure, chemical degradation and bioactivity of glasses in the CaO-MgO-SiO2-P2O5-CaF2 system. The (29)Si and (31)P magic angle spinning-nuclear magnetic resonance spectroscopy of melt-quenched glasses with varying Na2O/MgO ratios exhibit a silicate glass network with the dominance of Q(2)(Si) units and phosphorus mainly forming orthophosphate species. Sodium incorporation in the glasses did not induce a significant structural change in the silicate network, while it did influence the phosphate environment due to its lower ionic field strength in comparison with that of magnesium. The apatite forming ability of glasses has been investigated by immersion of glass powders in simulated body fluid (SBF) for time durations varying between 1h and 7 days while their chemical degradation has been studied in Tris-HCl in accordance with ISO-10993-14. Increasing Na(+)/Mg(2+) ratio caused a decrease in the chemical durability of glasses and in the apatite forming ability especially during initial steps of interaction between glass and SBF solution. The cellular responses were observed in vitro on bulk glass samples using mouse-derived pre-osteoblastic MC3T3-E1 cell line. The preliminary study suggested that the increasing alkali-concentration in glasses led to cytotoxicity in the cell culture medium.

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer.

Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

Differential stimulation of neurotrophin release by the biocompatible nano-material (carbon nanotube) in primary cultured neurons.

In order to develop novel, effective therapies for central nervous system regeneration, it is essential to better understand the role of neurotrophic factors and to design, accordingly, better artificial scaffolds to support both neurite outgrowth and synapse formation. Both nerve growth factor and brain-derived neurotrophic factor are major factors in neural survival, development, synaptogenesis, and synaptic connectivity of primary cultured neurons. As a prime candidate coating material for such neural cultures, carbon nanotubes offer unique structural, mechanical, and electrical properties. In this study, carbon nanotubes coated glass-coverslips were used as the matrix of a primary neural culture system used to investigate the effects of carbon nanotubes on neurite outgrowth and nerve growth factor/brain-derived neurotrophic factor release and expression. For these purposes, we performed comparative analyses of primary cultured neurons on carbon nanotubes coated, non-coated, and Matrigel-coated coverslips. The morphological findings showed definite carbon nanotubes effects on the neurite outgrowths and synaptogenic figures in both cortical and hippocampal neurons when compared with the non-coated negative control. Although the carbon nanotubes did not change neurotrophin expression levels, it stimulated brain-derived neurotrophic factor release into the media from both types of neurons. Accordingly, we suggest a different mechanism of action between carbon nanotubes and Matrigel in relation to the specific neurotrophic factors. Since carbon nanotubes supply long-term extracellular molecular cues for the survival and neurite outgrowths of cultured neurons, the results from this study will contribute to an understanding of carbon nanotubes biological effects and provide new insight into their role in the secretion of neurotrophic factors.

Gene delivery techniques for adult stem cell-based regenerative therapy.

Over the past decade, stem cells have been considered to be a promising resource to cure and regenerate damaged or diseased tissues with research extending from basic studies to clinical application. Furthermore, genetically modified stem cells have the potential to reduce tumorigenic risks and achieve safe tissue formation. Recent advances in genetic modification of stem cells have rendered these cells more accessible and stable. The successful genetic modification of stem cells relies heavily on designing vector systems, either viral or nonviral vectors, which can efficiently deliver therapeutic genes to the cells with minimum toxicity. Currently, viral vectors showing high transfection efficiencies still raise safety issues, whereas safer nonviral vectors exhibit extremely poor transfection in stem cells. Here, we attempt to review and discuss the main factors raising concern in previous reports, and devise strategies to solve the issues in gene delivery systems for successful stem cell-targeting regenerative therapy.

Expression profiles of subtracted mRNAs during cellular senescence in human mesenchymal stem cells derived from bone marrow.

Cellular senescence is an irreversible cell cycle arrest that limits the replicative lifespan of cells. Senescence suppresses development of tumors by regulating aging factors, such as cyclin dependent kinase inhibitor (CKI) and telomerase. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between young human mesenchymal stem cells (Y-hMSCs) and senescent human mesenchymal stem cells (S-hMSCs). We selected positive clones that were functionally characterized by referring to public databases using NCBI BLAST tool. This search revealed that 19 genes were downregulated, and 43 genes were upregulated in S-hMSCs relative to Y-hMSCs. Among subtracted clones in Y-hMSCs, most of genes markedly were related to metabolic functions. These genes, PDIA3, WDR1, FSTL1, COPG1, LMAN1, and PDIA6, significantly downregulated. Conversely, genes for subtracted clones in S-hMSCs were mostly associated with cell adhesion. In particular, the expression levels of 9 genes, HSP90B1, EID1, ATP2B4, DDAH1, PRNP, RAB1A, PGS5, TM4SF1 and SSR3, gradually increased during senescence. These genes have not previously been identified as being related to cellular senescence, but they seemed to be potentially affected during cellular senescence.

Isolation and characterization of novel, highly proliferative human CD34/CD73-double-positive testis-derived stem cells for cell therapy.

Human adult stem cells are a readily available multipotent cell source that can be used in regenerative medicine. Despite many advantages, including low tumorigenicity, their rapid senescence and limited plasticity have curtailed their use in cell-based therapies. In this study, we isolated CD34/CD73-double-positive (CD34(+)/CD73(+)) testicular stromal cells (HTSCs) and found that the expression of CD34 was closely related to the cells' stemness and proliferation. The CD34(+)/CD73(+) cells grew in vitro for an extended period of time, yielding a multitude of cells (5.6×10(16) cells) without forming tumors in vivo. They also differentiated into all three germ layer lineages both in vitro and in vivo, produced cartilage more efficiently compared to bone marrow stem cells and, importantly, restored erectile function in a cavernous nerve crush injury rat model. Thus, these HTSCs may represent a promising new autologous cell source for clinical use.

Bone tissue engineering of induced pluripotent stem cells cultured with macrochanneled polymer scaffold.

A reliable source of osteogenic cells is an essential factor for bone tissue engineering. In this study, human-induced pluripotent stem cells (hiPSCs) without an embryoid body step were cultured in macrochanneled poly(caprolactone) (PCL) scaffolds prepared using a robotic dispensing technique, after which osteogenesis was promoted by the addition of exogenous osteogenic factors. The osteogenesis of the hiPSCs was demonstrated based on the detection of osteogenic molecules, such as osteopontin, using flow cytometry analysis, quantitative polymerase chain reaction and western blotting. Thereafter, the cell-scaffold constructs were transplanted into the subcutaneous site of male athymic mice. At 4 weeks after implantation, histological assays (hematoxylin & eosin staining, Alizarin red staining, and osteocalcin immunostaining) were conducted to determine the bone induction of hiPSCs. The results indicated a production of pronounced levels of extracellular matrices and their mineral deposition within the cell-scaffold implant, suggesting possible in vivo bone induction by the hiPSCs-based tissue engineering approach. The results presented here provide useful information regarding the tissue engineering of bone utilizing hiPSCs in conjunction with cell-supporting scaffolds.

Robocasting nanocomposite scaffolds of poly(caprolactone)/hydroxyapatite incorporating modified carbon nanotubes for hard tissue reconstruction.

Nanocomposite scaffolds with tailored 3D pore configuration are promising candidates for the reconstruction of bone. Here we fabricated novel nanocomposite bone scaffolds through robocasting. Poly(caprolactone) (PCL)-hydroxyapatite (HA) slurry containing ionically modified carbon nanotubes (imCNTs) was robotic-dispensed and structured layer-by-layer into macrochanneled 3D scaffolds under adjusted processing conditions. Homogeneous dispersion of imCNTs (0.2 wt % relative to PCL-HA) was achieved in acetone, aiding in the preparation of PCL-HA-imCNTs slurry with good mixing property. Incorporation of imCNTs into PCL-HA composition significantly improved the compressive strength and elastic modulus of the robotic-dispensed scaffolds (~1.5-fold in strength and ~2.5-fold in elastic modulus). When incubated in simulated body fluid (SBF), PCL-HA-imCNT nanocomposite scaffold induced substantial mineralization of apatite in a similar manner to the PCL-HA scaffold, which was contrasted in pure PCL scaffold. MC3T3-E1 cell culture on the scaffolds demonstrated that cell proliferation levels were significantly higher in both PCL-HA-imCNT and PCL-HA than in pure PCL, and no significant difference was found between the nanocomposite scaffolds. When the PCL-HA-imCNT scaffold was implanted into a rat subcutaneous tissue for 4 weeks, soft fibrous tissues with neo-blood vessels formed well in the pore channels of the scaffolds without any significant inflammatory signs. Tissue reactions in PCL-HA-imCNT scaffold were similar to those in PCL-HA scaffold, suggesting incorporated imCNT did not negate the beneficial biological roles of HA. While more long-term in vivo research in bone defect models is needed to confirm clinical availability, our results suggest robotic-dispensed PCL-HA-imCNT nanocomposite scaffolds can be considered promising new candidate matrices for bone regeneration.

Performance of evacuated calcium phosphate microcarriers loaded with mesenchymal stem cells within a rat calvarium defect.

Tissue engineering of stem cells in concert with 3-dimensional (3D) scaffolds is a promising approach for regeneration of bone tissues. Bioactive ceramic microspheres are considered effective 3D stem cell carriers for bone tissue engineering. Here we used evacuated calcium phosphate (CaP) microspheres as the carrier of mesenchymal stem cells (MSCs) derived from rat bone marrow. The performance of the CaP-MSCs construct in bone formation within a rat calvarium defect was evaluated. MSCs were first cultured in combination with the evacuated microcarriers for 7 days in an osteogenic medium, which was then implanted in the 6 mm-diameter calvarium defect for 12 weeks. For comparison purposes, a control defect and cell-free CaP microspheres were also evaluated. The osteogenic differentiation of MSCs cultivated in the evacuated CaP microcarriers was confirmed by alkaline phosphatase staining and real time polymerase chain reaction. The in vivo results confirmed the highest bone formation was attained in the CaP microcarriers combined with MSCs, based on microcomputed tomography and histological assays. The results suggest that evacuated CaP microspheres have the potential to be useful as stem cell carriers for bone tissue engineering.

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells.

Undifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.