Gut Mucosal Microbiome Is Perturbed in Rheumatoid Arthritis Mice and Partly Restored after TDAG8 Deficiency or Suppression by Salicylanilide Derivative.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by chronic joint inflammation and pain. We previously found that the deletion of T-cell death-associated gene 8 (TDAG8) significantly reduces disease severity and pain in RA mice. Whether it is by modulating gut microbiota remains unclear. In this study, 64 intestinal samples of feces, cecal content, and cecal mucus from the complete Freund's adjuvant-induced arthritis mouse models were compared. The α- and ß-diversity indices of the microbiome were significantly lower in RA mice. Cecal mucus showed a higher ratio of Firmicutes to Bacteroidetes in RA than healthy mice, suggesting the ratio could serve as an RA indicator. Four core genera, Eubacterium_Ventriosum, Alloprevotella, Rikenella, and Treponema, were reduced in content in both feces and mucus RA samples, and could serve microbial markers representing RA progression. TDAG8 deficiency decreased the abundance of proinflammation-related Eubacterium_Xylanophilum, Clostridia, Ruminococcus, Paraprevotella, and Rikenellaceae, which reduced local mucosal inflammation to relieve RA disease severity and pain. The pharmacological block of the TDAG8 function by a salicylanilide derivative partly restored the RA microbiome to a healthy composition. These findings provide a further understanding of specific bacteria interactions with host gut mucus in the RA model. The modulation by TDAG8 on particular bacteria can facilitate microbiota-based therapy.

Miniaturized Biomedical Sensors for Enumeration of Extracellular Vesicles.

In this research, we have realized a rapid extracellular vesicle (EV) quantification methodology using a high field modulated AlGaN/GaN high electron mobility (HEMT) biosensor. The unique sensing structure facilitated the detection of the sub-cellular components in physiological salt environment without requiring extensive sample pre-treatments. The high field operation of GaN HEMT biosensor provides high sensitivity and wide dynamic range of detection of EVs (107⁻1010 EVs/mL). An antibody specific to the known surface marker on the EV was used to capture them for quantification using an HEMT biosensor. Fluorescence microscopy images confirm the successful capture of EVs from the test solution. The present method can detect EVs in high ionic strength solution, with a short sample incubation period of 5 min, and does not require labels or additional reagents or wash/block steps. This methodology has the potential to be used in clinical applications for rapid EV quantification from blood or serum for the development of diagnostic and prognostic tools.

Digitized Hand Skateboard Based on IR-Camera for Upper Limb Rehabilitation.

Abnormal upper limb function seriously impacts a patient's daily life. After receiving emergency treatment patient should receive function-rebuilding and recovery training. The objective of this study is to integrate IR-camera, an infrared emitter, with a conventional passive hand skateboard training device for conventional upper limb training and the training process is comprehensively recorded and analyzed. Patients participating in the occupational therapy have a binding band attached to hand skateboard on the table to guide the patient in moving the hand skateboard along the designated path to train the patient's upper limbs. Six people with normal upper limb function participated in the stability test. The device repeatability and test results were verified acceptable. Eight patients with abnormal upper limb function (their upper limb function was damaged due to stroke, MMSE > =27) were trained for 4 weeks. The patient scores in finishing rate and finishing time showed significant improvement. The paired T test results (satisfy p < 0.05 or p < 0.01) between wk-1 and wk-2 are significant. The paired T test results (satisfy p < 0.01) between wk-1 and wk-4 are extremely significant. The new IR-Camera system focuses continuously on the "Figure of eight" curve. The system is light weight and convenient for stroke in home use. The study applies IR-camera technology to the conventional hand skateboard for upper limb training. The experiments show that the hardware of the proposed device no longer delays in response and can result in obvious clinical advances. The proposed device is verified worthy of promotion.

A PDMS-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation.

The conventional hanging drop technique is the most widely used method for embryoid body (EB) formation. However, this method is labor intensive and limited by the difficulty in exchanging the medium. Here, we report a microfluidic chip-based approach for high-throughput formation of EBs. The device consists of microfluidic channels with 6 × 12 opening wells in PDMS supported by a glass substrate. The PDMS channels were fabricated by replicating polydimethyl-siloxane (PDMS) from SU-8 mold. The droplet formation in the chip was tested with different hydrostatic pressures to obtain optimal operation pressures for the wells with 1000 µm diameter openings. The droplets formed at the opening wells were used to culture mouse embryonic stem cells which could subsequently developed into EBs in the hanging droplets. This device also allows for medium exchange of the hanging droplets making it possible to perform immunochemistry staining and characterize EBs on chip.

Improvement in the physiological function and standing stability based on kinect multimedia for older people.

[Purpose] The increase in the Taiwanese older population is associated with age-related inconveniences. Finding adequate and simple physical activities to help the older people maintaining their physiological function and preventing them from falls has become an urgent social issue. [Subjects and Methods] This study aimed to design a virtual exercise training game suitable for Taiwanese older people. This system will allow for the maintenance of the physiological function and standing stability through physical exercise, while using a virtual reality game. The participants can easily exercise in a carefree, interactive environment. This study will use Kinect for Windows for physical movement detection and Unity software for virtual world development. [Results] Group A and B subjects were involved in the exercise training method of Kinect interactive multimedia for 12 weeks. The results showed that the functional reach test and the unipedal stance test improved significantly. [Conclusion] The physiological function and standing stability of the group A subjects were examined at six weeks post training. The results showed that these parameters remained constant. This proved that the proposed system provide substantial support toward the preservation of the Taiwanese older people' physiological function and standing stability.

Multimedia virtualized environment for shoulder pain rehabilitation.

[Purpose] Researchers imported games and virtual reality training to help participants train their shoulders in a relaxed environment. [Subjects and Methods] This study included the use of Kinect somatosensory device with Unity software to develop 3-dimensional situational games. The data collected from this training process can be uploaded via the Internet to a cloud or server for participants to perform self-inspection. The data can be a reference for the medical staff to assess training effectiveness for those with impairments and plan patient rehabilitation courses. [Results] In the training activities, 8 subjects with normal shoulder function demonstrated that the system has good stability and reproducibility. Six subjects with impaired shoulder underwent 6 weeks of training. During the third week of training, average performance stabilized. The t-test comparing 1-2 weeks to 3-4 weeks and 5-6 weeks showed significant differences. [Conclusion] Using games as training methods improved patient concentration, interest in participation and allowed patients to forget about their body discomfort. The equipment utilized in this study is inexpensive, easy to obtain, and the system is easy to install. People can perform simple self-training both at home or in the office.

Application of RFID technology-upper extremity rehabilitation training.

[Purpose] Upper extremity rehabilitation after an injury is very important. This study proposes radio frequency identification (RFID) technology to improve and enhance the effectiveness of the upper extremity rehabilitation. [Subjects and Methods] People use their upper extremities to conduct daily activities. When recovering from injuries, many patients neglect the importance of rehabilitation, which results in degraded function. This study recorded the training process using the traditional rehabilitation hand gliding cart with a RFID reader, RFID tags in the panel, and a servo host computer. [Results] Clinical evidence, time taken to achieve a full score, counts of missing the specified spots, and Brunnstrom stage of aided recovery, the proximal part of the upper extremity show that the RFID-based upper extremity training significantly and reduce negative impacts of the disability in daily life and activities. [Conclusion] This study combined a hand-gliding cart with an RFID reader, and when patients moved the cart, the movement could be observed via the activated RFID tags. The training data was collected and quantified for a better understanding of the recovery status of the patients. Each of the participating patients made progress as expected.

Application of the Blobo bluetooth ball in wrist rehabilitation training.

[Purpose] The introduction of emerging technologies such as the wireless Blobo bluetooth ball with multimedia features can enhance wrist physical therapy training, making it more fun and enhancing its effects. [Methods] Wrist injuries caused by fatigue at work, improper exercise, and other conditions are very common. Therefore, the reconstruction of wrist joint function is an important issue. The efficacy of a newly developed integrated wrist joint rehabilitation game using a Blobo bluetooth ball with C# software installed was tested in wrist rehabilitation (Flexion, Extension, Ulnar Deviation, Radial Deviation). [Results] Eight subjects with normal wrist function participated in a test of the system's stability and repeatability. After performing the Blobo bluetooth ball wrist physical therapy training, eight patients with wrist dysfunction experienced approximately 10° improvements in range of motion (ROM) of flexion extension, and ulnar deviation and about 6° ROM improvement in radial deviation. The subjects showed progress in important indicators of wrist function. [Conclusion] This study used the Blobo bluetooth ball in wrist physical therapy training and the preliminary results were encouraging. In the future, more diverse wrist or limb rehabilitation games should be developed to meet the needs of physical therapy training.

Balance training using an interactive game to enhance the use of the affected side after stroke.

[Purpose] Stroke and other cerebrovascular diseases are major causes of adult mobility problems. Because stroke immobilizes the affected body part, balance training uses the healthy body part to complete the target movement. The muscle utilization rate on the stroke affected side is often reduced which further hinders affected side functional recovery in rehabilitation. [Subjects and Methods] This study tested a newly-developed interactive device with two force plates to measuring right and left side centers of pressure, to establish its efficacy in the improvement of the static standing ability of patients with hemiplegia. An interactive virtual reality game with different side reaction ratios was used to improve patient balance. The feasibility of the proposed approach was experimentally demonstrated. [Results] Although the non-affected-side is usually used to support the body weight in the standing position, under certain circumstances the patients could switch to using the affected side. A dramatic improvement in static standing balance control was achieved in the eyes open condition. [Conclusion] The proposed dual force plate technique used in this study separately measured the affected and non-affected-side centers of pressure. Based on this approach, different side ratio integration was achieved using an interactive game that helped stroke patients improve balance on the affected side. Only the patient who had suffered stroke relatively recently benefited significantly. The proposed technique is of little benefit for patients whose mobility has stagnated to a certain level.

Investigation of C-terminal domain of SARS nucleocapsid protein-Duplex DNA interaction using transistors and binding-site models.

AlGaN/GaN high electron mobility transistors (HEMTs) were used to sense the binding between double stranded DNA (dsDNA) and the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (N protein). The sensing signals were the drain current change of the HEMTs induced by the protein-dsDNA binding. Binding-site models using surface coverage ratios were utilized to analyze the signals from the HEMT-based sensors to extract the dissociation constants and predict the number of binding sites. Two dissociation constants, K D1 = 0.0955 nM, K D2 = 51.23 nM, were obtained by fitting the experimental results into the two-binding-site model. The result shows that this technique is more competitive than isotope-labeling electrophoretic mobility shift assay (EMSA). We demonstrated that AlGaN/GaN HEMTs were highly potential in constructing a semiconductor-based-sensor binding assay to extract the dissociation constants of nucleotide-protein interaction.

Risk Factors for Unplanned Early Implantable Port Catheter Removal in Adult Hematology Cancer Patients Receiving Chemotherapy: A Propensity Score Matching Study.

Purpose: Implantable port catheter is a reliable vascular access for chemotherapy infusion in cancer patients. However, patients with hematology malignancies usually present with a myriad of blood cell abnormalities that put them at risk of infection and mechanical problems requiring catheter removal. This study aims to determine the risk factors associated with unplanned (catheter removal other than completion of treatment plan) early (within 90 days of catheter implantation) implantable port catheter removal. Patients and Methods: A retrospective, propensity score-matched study of 386 patients with hematology malignancies who received implantable venous access ports between January 2015 and December 2022. We conducted a univariate analysis to select the variables for propensity score matching. Patients with unplanned early implantable port catheter removal (early group) were matched 1:1 to patients without unplanned early removal (non-early group). Results: Univariate analysis demonstrated a statistically significant difference between early and non-early groups for age (p = 0.048), hemoglobin level (p = 0.028), thrombocytopenia (p = 0.025), and PG-SGA (p < 0.001). Thrombocytopenia was the only independent risk factor with a statistically significant difference in Cox proportional hazard analysis, HR 2.823, 95 CI 1.050-7.589, p = 0.040. The median catheter survival for patients with thrombocytopenia was 61 days (95% CI 28.58-93.42) compared to 150 days (95% CI 9.81-290.19) for patients without thrombocytopenia, p = 0.015. Patient survival is not affected by early catheter removal. The median survival for patients in the early group was 28.28 months (95% CI 27.43-29.15) compared to 32.39 months (95% CI 24.11-40.68), for the non-early group, p = 0.709. Conclusion: Hematology malignancy patients with thrombocytopenia are at high risk for unplanned early port catheter removal without survival difference.

The Epidemiology, Management and Therapeutic Outcomes of Subdural Empyema in Neonates with Acute Bacterial Meningitis.

Background: Subdural empyema is one of the more serious complications of bacterial meningitis and therapeutic challenges to clinicians. We aimed to evaluate the clinical characteristics, treatment, and outcome of subdural empyema in neonates with bacterial meningitis. Methods: A retrospective cohort study was conducted in two medical centers in Taiwan that enrolled all cases of neonates with subdural empyema after bacterial meningitis between 2003 and 2020. Results: Subdural empyema was diagnosed in 27 of 153 (17.6%) neonates with acute bacterial meningitis compared with cases of meningitis without subdural empyema. The demographics and pathogen distributions were comparable between the study group and the controls, but neonates with subdural empyema were significantly more likely to have clinical manifestations of fever (85.2%) and seizure (81.5%) (both p values < 0.05). The cerebrospinal fluid results of neonates with subdural empyema showed significantly higher white blood cell counts, lower glucose levels and higher protein levels (p = 0.011, 0.003 and 0.006, respectively). Neonates with subdural empyema had a significantly higher rate of neurological complications, especially subdural effusions and periventricular leukomalacia. Although the final mortality rate was not increased in neonates with subdural empyema when compared with the controls, they were often treated much longer and had a high rate of long-term neurological sequelae. Conclusions: Subdural empyema is not uncommon in neonates with acute bacterial meningitis and was associated with a high risk of neurological complications, although it does not significantly increase the final mortality rate. Close monitoring of the occurrence of subdural empyema is required, and appropriate long-term antibiotic treatment after surgical intervention may lead to optimized outcomes.

Identifying and characterizing Yarrowia keelungensis sp. nov., an oil-degrading yeast isolated from the sea surface microlayer.

Ascomycetous yeast strain SM-22 was isolated from the sea-surface microlayer near the Keelung City off the northern coast of Taiwan. This strain showed a cell surface hydrophobicity higher than 90 %, moderate UV A/B resistance, and it degraded 68 % of the total petroleum hydrocarbon content of an artificial seawater medium containing 1 % (v v(-1)) diesel oil within 15 days at 25 °C. The closest phylogenetic relative of this strain is Candida oslonensis CBS 10146(T), but it differs from strain SM-22 by a 3.7 % divergence (including 18 nucleotide substitutions and 2 gaps) in the D1/D2 domain sequence of the large subunit rRNA gene. This difference clearly suggests that the strain SM-22 represents a distinct species. Strain SM-22 does not produce ascospores on common sporulation media and it can therefore be considered an anamorph of the genus Yarrowia. Thus, the name Yarrowia keelungensis sp. nov. (type strain SM-22(T) = BCRC 23110(T) = JCM 14894(T) = CBS 11062(T)) is proposed as a novel species of genus Yarrowia.

The Clinical Characteristics, Microbiology and Risk Factors for Adverse Outcomes in Neonates with Gram-Negative Bacillary Meningitis.

Background: We aimed to describe the clinical features of Gram-negative bacillary (GNB) meningitis in neonates and investigate the risk factors associated with final adverse outcomes of neonatal GNB meningitis. Methods: From 2003 to 2020, all neonates (aged ≤ 90 days old) with bacterial meningitis who were hospitalized in four tertiary-level neonatal intensive care units (NICUs) of two medical centers in Taiwan were enrolled. Neonates with GNB meningitis were compared with those with Streptococcus agalactiae (group B streptococcus, GBS) meningitis. Results: During the study period, a total of 153 neonates with bacterial meningitis were identified and enrolled. GNB and GBS accounted for 40.5% (n = 62) and 35.3% (n = 54) of all neonatal bacterial meningitis, respectively. In neonates with GNB meningitis, the final mortality rate was 6.5% (4 neonates died); 48 (77.4%) had neurological complications, and 26 (44.8%) of 58 survivors had neurological sequelae at discharge. Although the final outcomes were comparable between neonates with GNB meningitis and those with GBS meningitis, neonates with GNB meningitis were more likely to have more severe clinical manifestations initially and have ventriculomegaly at follow-up. After multivariate logistic regression analysis, neonates with seizure at onset, early onset sepsis, and requirement of surgical intervention for neurological complications were independently associated with final adverse outcomes. Conclusions: GNB meningitis was associated with a high risk of neurological complications and sequelae, although it did not significantly increase the final mortality rate. Close monitoring of the occurrence of neurological complications and advanced therapeutic strategies to optimize the outcomes are urgently needed in the future.

Influences of Initial Empiric Antibiotics with Ampicillin plus Cefotaxime on the Outcomes of Neonates with Respiratory Failure: A Propensity Score Matched Analysis.

Background: Empiric antibiotics are often prescribed in critically ill and preterm neonates at birth until sepsis can be ruled out. Although the current guideline suggests narrow-spectrum antibiotics, an upgrade in antibiotics is common in the neonatal intensive care unit. The impacts of initial broad-spectrum antibiotics on the outcomes of critically ill neonates with respiratory failure requiring mechanical intubation have not been well studied. Methods: A total of 1162 neonates from a tertiary level neonatal intensive care unit (NICU) in Taiwan who were on mechanical ventilation for respiratory distress/failure at birth were enrolled, and neonates receiving ampicillin plus cefotaxime were compared with those receiving ampicillin plus gentamicin. Propensity score-matched analysis was used to investigate the effects of ampicillin plus cefotaxime on the outcomes of critically ill neonates. Results: Ampicillin plus cefotaxime was more frequently prescribed for intubated neonates with lower birth weight, higher severity of illness, and those with a high risk of early-onset sepsis. Only 11.1% of these neonates had blood culture-confirmed early-onset sepsis and/or congenital pneumonia. The use of ampicillin plus cefotaxime did not significantly contribute to improved outcomes among neonates with early-onset sepsis. After propensity score-matched analyses, the critically ill neonates receiving ampicillin plus cefotaxime had significantly worse outcomes than those receiving ampicillin plus gentamicin, including a higher risk of late-onset sepsis caused by multidrug-resistant pathogens (11.2% versus 7.1%, p = 0.027), longer duration of hospitalization (median [IQR], 86.5 [47-118.8] days versus 78 [45.0-106.0] days, p = 0.002), and a significantly higher risk of in-hospital mortality (14.2% versus 9.6%, p = 0.023). Conclusions: Ampicillin plus cefotaxime should not be routinely prescribed as the empiric antibiotics for critically ill neonates at birth because they were associated with a higher risk of infections caused by multidrug-resistant pathogens and final worse outcomes.

Combinatory effects of current regimens and Guizhi Fuling Wan on the development of endometriosis.

OBJECTIVE: Endometriosis, defined as the growth of endometrial glands and stromal cells in a heterotopic location under the cyclic influence of ovarian hormones, is a common gynecological disorder manifested by chronic pelvic pain and infertility. In traditional Chinese medicine, endometriosis is characterized by stagnation of vital energy (qi) and blood stasis. Guizhi Fuling Wan (GFW) was first described in Chinese canonical medicine to treat disorders associated with stagnation of qi and blood stasis, including endometriosis. Therefore, the current study aimed to test the effects of combining GFW with western medicine on the suppression of endometriosis. MATERIALS AND METHODS: Endometriosis was generated by suturing endometrial tissue on the peritoneal wall of C57BL/6JNarl mice. The mice were subsequently treated with either GFW or current hormonal therapies or in combination for 28 days. RESULTS: Endometriosis development was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone or GFW + medroxyprogesterone acetate (MPA). The expression of intercellular adhesion molecule 1 (ICAM-1) was inhibited by GFW, Gestrinone, MPA, Visanne, GFW + Gestrinone, GFW + MPA and GFW + Visanne. Vascular endothelial growth factor (VEGF) expression was inhibited by GFW, Gestrinone, Visanne, GFW + Gestrinone and GFW + MPA. Both ICAM-1- and VEGF-reducing effects of GFW were attenuated by western medicines. Administration of GFW, MPA, Visanne, GFW + MPA and GFW + Visanne also correspondingly reduced macrophage population in peritoneal fluid. GFW, MPA, Visanne, GFW + MPA and GFW + Visanne enhanced B-cell population in peritoneal fluid. CONCLUSION: The current study reveals the therapeutic effects of GFW on endometriosis. However, the combination of GFW and current hormonal therapies potentially impedes the efficacy of each individual agent in treating endometriosis.

A Microfluidic Flip-Chip Combining Hydrodynamic Trapping and Gravitational Sedimentation for Cell Pairing and Fusion.

Cancer cell-immune cell hybrids and cancer immunotherapy have attracted much attention in recent years. The design of efficient cell pairing and fusion chips for hybridoma generation has been, subsequently, a subject of great interest. Here, we report a three-layered integrated Microfluidic Flip-Chip (MFC) consisting of a thin through-hole membrane sandwiched between a mirrored array of microfluidic channels and saw-tooth shaped titanium electrodes on the glass. We discuss the design and operation of MFC and show its applicability for cell fusion. The proposed device combines passive hydrodynamic phenomenon and gravitational sedimentation, which allows the transportation and trapping of homotypic and heterotypic cells in large numbers with pairing efficiencies of 75~78% and fusion efficiencies of 73%. Additionally, we also report properties of fused cells from cell biology perspectives, including combined fluorescence-labeled intracellular materials from THP1 and A549, mixed cell morphology, and cell viability. The MFC can be tuned for pairing and fusion of cells with a similar protocol for different cell types. The MFC can be easily disconnected from the test setup for further analysis.

Implementation of interactive games to a shoulder rehabilitation and evaluation system.

BACKGROUND: After an operation, the shoulder and wrist might not be able to lift and swing freely, and must be assisted with rehabilitation training. OBJECTIVE: In this paper, Kinect combined with multiple sensors of a Bluetooth ball is proposed to improve the measurement function of the arm's micro-motion trajectory, rotation amount, and acceleration, which cannot be detected by Kinect alone. METHODS: We designed two virtual scene rehabilitation games for clinical trials. We performed validity analysis with a paired sample t-test. RESULTS: A significance value of P*< 1 was obtained, and the arm lift angle shows an improvement from 30∘ to 60∘, indicating that the range of motion of the hand and shoulder is gradually improving. CONCLUSION: Experiments show that virtual games combined with multiple sensors can better understand the patient's rehabilitation situation.

Diverse Community Structures in the Neuronal-Level Connectome of the Drosophila Brain.

Drosophila melanogaster is one of the most important model animals in neurobiology owing to its manageable brain size, complex behaviour, and extensive genetic tools. However, without a comprehensive map of the brain-wide neural network, our ability to investigate brain functions at the systems level is seriously limited. In this study, we constructed a neuron-to-neuron network of the Drosophila brain based on the 28,573 fluorescence images of single neurons in the newly released FlyCircuit v1.2 (http://www.flycircuit.tw) database. By performing modularity and centrality analyses, we identified eight communities (right olfaction, left olfaction, olfactory core, auditory, motor, pre-motor, left vision, and right vision) in the brain-wide network. Further investigation on information exchange and structural stability revealed that the communities of different functions dominated different types of centralities, suggesting a correlation between functions and network structures. Except for the two olfaction and the motor communities, the network is characterized by overall small-worldness. A rich club (RC) structure was also found in this network, and most of the innermost RC members innervated the central complex, indicating its role in information integration. We further identified numerous loops with length smaller than seven neurons. The observation suggested unique characteristics in the information processing inside the fruit fly brain.

Cell migration of preosteoblast cells on a clinical gelatin sponge for 3D bone tissue engineering.

Using three-dimensional (3D) bone engineering to fabricate bone segments is a better choice for repairing bone defects than using autologous bone. However, biomaterials for bone engineering are burdened with some clinical safety concerns. In this study, we layered commonly found clinical materials, hemostatic gelatin sponges, in a novel manner to create a 3D scaffold for bone engineering purposes. We further examined the comparable benefits of our design with both closed- and open-bottom holders. Cells in stacked layer disc systems were examined after a week of growth and differentiation. Osteoblasts in the outer layers of both closed- and open-bottom holder systems displayed gradually increased alkaline phosphatase (ALP) activity but decreased osteopontin (OPN) expression. Further, cell proliferation assays and LIVE/DEAD staining revealed decreased viable cell counts in the top layer with increased incubation time. However, while layered disc systems with closed-bottom holders underwent differentiation, they kept more differentiated cells alive within the gelatin sponge disc scaffold after 28 d of culturing. Whether cells were inoculated into the top, middle, or bottom portions of the layered disc stack, osteoblasts showed a preference for migrating to the top layer, in keeping with the oxygen and nutrients gradients. Regarding practical application, this study offers valuable information to promote the use of hemostatic gelatin sponges for bone engineering.