Delayed revascularization of islets after transplantation by IL-6 blockade in pig to non-human primate islet xenotransplantation model.

BACKGROUND: Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL-6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL-6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non-human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined. METHODS: Pig islets were isolated from designated pathogen-free (DPF) SNU miniature pigs and transplanted via portal vein into five streptozotocin-induced diabetic monkeys. One group (n = 2, basal group) was treated with anti-thymoglobulin (ATG), anti-CD40 antibody (2C10R4), sirolimus, and tacrolimus, and the other group was additionally given tocilizumab on top of basal immunosuppression (n = 3, Tocilizumab group). To confirm IL-6 blocking effect, C-reactive protein (CRP) levels and serum IL-6 concentration were measured. Scheduled biopsy of the margin of the posterior segment right lobe inferior of the liver was performed at 3 weeks after transplantation to assess the degree of revascularization of the transplanted islets. Immunohistochemical staining using anti-insulin, anti-CD31 antibodies, and lectin IB4 was conducted to find the origin of endothelial cells in the islet graft. RESULTS: CRP significantly increased at 1~2 days after transplantation in Basal group, but not in Tocilizumab group, and higher serum IL-6 concentration was measured in latter group, showing the biological potency of tocilizumab. In Basal group, well-developed endothelial cells were observed on the peri- and intraislet area, whereas the number of CD31+ cells in the intraislet space was significantly reduced in Tocilizumab group. Finally, new endothelial cells in the pig islet graft were positive for CD31, but not for lectin IB4, suggesting that they are originated from the recipient monkey. CONCLUSIONS: Our results demonstrated that tocilizumab can delay revascularization of the transplanted islet, although this effect had no significant correlation to the overall islet graft survival. In the pig to NHP islet xenotransplantation model, the endothelial cells from recipient monkey form new blood vessels in and around pig islets.

Electrothermal MEMS parallel plate rotation for single-imager stereoscopic endoscopes.

This work reports electrothermal MEMS parallel plate-rotation (PPR) for a single-imager based stereoscopic endoscope. A thin optical plate was directly connected to an electrothermal MEMS microactuator with bimorph structures of thin silicon and aluminum layers. The fabricated MEMS PPR device precisely rotates an transparent optical plate up to 37° prior to an endoscopic camera and creates the binocular disparities, comparable to those from binocular cameras with a baseline distance over 100 µm. The anaglyph 3D images and disparity maps were successfully achieved by extracting the local binocular disparities from two optical images captured at the relative positions. The physical volume of MEMS PPR is well fit in 3.4 mm x 3.3 mm x 1 mm. This method provides a new direction for compact stereoscopic 3D endoscopic imaging systems.

Beneficial effects of fresh and fermented kimchi in prediabetic individuals.

BACKGROUND: With the increased incidence of diabetes mellitus, the importance of early intervention in prediabetes has been emphasized. We previously reported that fermented kimchi, a traditional Korean food, reduced body weight and improved metabolic factors in overweight participants. We hypothesized that kimchi and its fermented form would have beneficial effects on glucose metabolism in patients with prediabetes. METHODS: A total of 21 participants with prediabetes were enrolled. During the first 8 weeks, they consumed either fresh (1-day-old) or fermented (10-day-old) kimchi. After a 4-week washout period, they switched to the other type of kimchi for the next 8 weeks. RESULTS: Consumption of both types of kimchi significantly decreased body weight, body mass index, and waist circumference. Fermented kimchi decreased insulin resistance, and increased insulin sensitivity, QUICKI and disposition index values (p = 0.004 and 0.028, respectively). Systolic and diastolic blood pressure (BP) decreased significantly in the fermented kimchi group. The percentages of participants who showed improved glucose tolerance were 9.5 and 33.3% in the fresh and fermented kimchi groups, respectively. CONCLUSIONS: Consumption of kimchi had beneficial effects on glucose metabolism-related and anthropometric factors in participants with prediabetes. Fermented kimchi had additional effects on BP and insulin resistance/sensitivity. The percentage of participants who showed improvement in glucose tolerance was high in the fermented kimchi group.

Sleep duration and glycemic control in patients with diabetes mellitus: Korea National Health and Nutrition Examination Survey 2007-2010.

Short sleep duration has been reported to increase the risk of diabetes. However, the influence of sleep duration on glycemic control in diabetic patients has not been clarified. In this study we evaluated the association between sleep duration and glycemic control in diabetic patients. We analyzed the data from the Korea National Health and Nutrition Examination Survey (KNHANES) 2007-2010. Sleep duration was classified into five groups: <6, 6, 7, 8, and ≥9 h/day. Fasting blood glucose and HbA1c showed a U-shaped trend according to sleep duration. Sleep duration of 7 h/day had the lowest HbA1c (7.26%) among the subjects (P=0.026). In the older age group (≥65 yr), a sleep duration of 6 h/day was associated with the lowest HbA1c (7.26%). The adjusted odds ratio (OR) with a 95% confidence interval (CI) of worse glycemic control (HbA1c ≥7.0%) in group of sleep duration of ≥9 h/day was 1.48 (1.04-2.13) compared with the group of 7 h/day. This relationship disappeared after adjusting duration of diabetes (OR, 1.38; 95% CI, 0.93-2.03). Our results suggest that sleep duration and glycemic control in diabetic patients has U-shaped relationship which was mainly affected by duration of diabetes.

Rationally designed bioactive milk-derived protein scaffolds enhanced new bone formation.

Recently, a number of studies reported that casein was composed of various multifunctional bioactive peptides such as casein phosphopeptide and ß-casochemotide-1 that bind calcium ions and induce macrophage chemotaxis, which is crucial for bone homeostasis and bone fracture repair by cytokines secreted in the process. We hypothesized that the effects of the multifunctional biopeptides in casein would contribute to improving bone regeneration. Thus, we designed a tissue engineering platform that consisted of casein and polyvinyl alcohol, which was a physical-crosslinked scaffold (milk-derived protein; MDP), via simple freeze-thaw cycles and performed surface modification using 3,4-dihydroxy-l-phenylalanine (DOPA), a mussel adhesive protein, for immobilizing adhesive proteins and cytokines for recruiting cells in vivo (MDP-DOPA). Both the MDP and MDP-DOPA groups proved indirectly contribution of macrophages migration as RAW 264.7 cells were highly migrated toward materials by contained bioactive peptides. We implanted MDP and MDP-DOPA in a mouse calvarial defect orthotopic model and evaluated whether MDP-DOPA showed much faster mineral deposition and higher bone density than that of the no-treatment and MDP groups. The MDP-DOPA group showed the accumulation of host M2 macrophages and mesenchymal stem cells (MSCs) around the scaffold, whereas MDP presented mostly M1 macrophages in the early stage.

Fibrinogen-based cell and spheroid sheets manipulating and delivery for mouse hindlimb ischemia.

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higherin vitrotubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research.

Scalable Anatomically-Tunable Fully In-Ear Dry-Electrode Array for User-Generic Unobtrusive Electrophysiology.

Traditional scalp EEG instrumentation is bulky and arduous to set up, requiring wires that constrain the subject's movement, conductive wet gels that dry over time which limits long-term recording, and/or is socially stigmatized. Therefore, there is growing research in in-ear EEG to increase user wearability, ease of use, and concealability. However, the fabrication of in-ear EEG sensors utilizes complex equipment and materials to capture the intricate geometry of the ear and to fabricate custom earpieces and electrodes. This work aims to lower the barrier of entry by decreasing the fabrication complexity by using PCB components with versatile, user-generic designs. Measured results on the assembled earpiece demonstrate that it viably captures eye blinks, jaw clench, auditory steady-state response (ASSR), and alpha modulation. Additionally, electrochemical impedance spectroscopy (EIS) experiments show reliable electrode-skin contact with impedance comparable to conventional dry-electrode designs at substantially greater channel density.

Enhanced extraction of skin interstitial fluid using a 3D printed device enabling tilted microneedle penetration.

Interstitial fluid (ISF) is a body fluid that fills, surrounds cells and contains various biomarkers, but it has been challenging to extract ISF in a reliable and sufficient amount with high speed. To address the issues, we developed the tilted microneedle ISF collecting system (TMICS) fabricated by 3D printing. In this system, the microneedle (MN) was inserted at 66° to the skin by TMICS so that the MN length could be extended within a safe range of skin penetration. Moreover, TMICS incorporating three MN patches created reliable ISF collecting conditions by penetrating the skin at consistent angle and force, 4.9 N. Due to the MN length increase and the patch number expansion, the surface area of the penetrated tissue was increased, thereby confirming that ISF extraction efficiency was improved. Skin ISF was collected into the paper reservoir on the patch, and the absorbed area was converted into a volume. ISF extraction from the rat skin in vivo by TMICS was well tolerated, and the 2.9 µL of ISF was obtained within 30 s. Therefore, TMICS is promising to apply in the diagnosis of multiple biomarkers in ISF with high speed and stability.

Antioxidant and anti-inflammatory activities of Prussian blue nanozyme promotes full-thickness skin wound healing.

Excessive reactive oxygen species (ROS) and unresolved inflammations are the major causes of impaired wound healing as they overwhelm the cellular antioxidant system and impede the healing process. In this study, we examined the application of Prussian blue (PB) nanozyme as a novel material for cutaneous wound healing through the alleviation of excessive ROS and inflammation modulation. The PB nanoparticles not only exhibited hydrogen peroxide (H2O2) degradation activity but also showed strong superoxide scavenging ability. PB nanozyme mitigated the intracellular ROS at a high oxidative stress environment, resulting in a pronounced cytoprotective effect. Moreover, PB nanozyme also displayed significant anti-inflammatory activity, as evident from the suppression of inflammatory mediators in the lipopolysaccharide (LPS) induced macrophage cells. Encouraged by the in vitro results, we evaluated the in vivo therapeutic efficacy of PB nanozyme in a full-thickness cutaneous wound model combined with LPS treatment to mimic bacterial infection. The beneficial effects of topically applied PB nanozyme on wound healing and tissue regeneration were evident compared to the control. The periodical administration of a low amount (50 µg × 4) of PB nanoparticles exhibited faster wound closure as well as collagen deposition, maturation, and organization. Moreover, the PB treatment effectively induced the differentiation of keratinocytes, enhanced the neovascularization, and reduced macrophage burden in the entire wound site. Thus, PB nanozyme not only accelerated the healing process in an infection-mimicking cutaneous wound model but also exhibited tissue regeneration characteristics owing to the synergistic effect of ROS-scavenging and anti-inflammatory activities.

Nanozyme Impregnated Mesenchymal Stem Cells for Hepatic Ischemia-Reperfusion Injury Alleviation.

Mesenchymal stem cell (MSC) based therapy holds great potential for treating numerous diseases owing to their capability to heal injured tissue/organs through paracrine factors secretion and immunomodulation. Despite the high hopes, the low viability of transplanted cells in the injured tissues due to the elevated oxidative stress levels remains the largest obstacle in MSC-based cell therapy. To achieve desired therapeutic efficiency, the survival of the transplanted MSCs in the high oxidative stress environment needs to be ensured. Herein, we proposed the use of a ROS-scavenging nanozyme to protect transplanted MSCs from oxidative stress-mediated apoptosis and thereby improve the therapeutic effect. Prussian blue (PB) nanoparticles as a biocompatible ROS-scavenging nanozyme were incorporated into the MSCs without affecting the stemness and differentiation potential of MSCs. The nanozyme impregnation significantly improved the survival of MSCs in a high oxidative stress condition as well as augmented their paracrine effect and anti-inflammatory properties, resulting in a profound therapeutic effect in vivo in the liver ischemia-reperfusion (I/R) injury animal model. Our results indicated that the nanozyme incorporation into MSCs is a simple but efficient way to improve the therapeutic potential of MSC-based cell therapy.

In situ pocket-type microcarrier (PMc) as a therapeutic composite: Regeneration of cartilage with stem cells, genes, and drugs.

We prepared pocket-type micro-carriers (PMc) with pores larger than 30 µm for use in cell delivery by adding 40 mg pluronic F-127 copolymers (F-127) to biodegradable PLGA dissolved in dichloromethane solution. The controlling the size of the pockets in this way facilitates the adhesion of cells by regulating the size of the pockets according to the cells having various sizes. The size of PMc pores could be controlled within a range of 2 to 30 µm by varying the F-127 content. The ratio of F-127 to DOPA-bPEI was most appropriate at 1: 1, and the pocket size at 10 mg/ml of F-127 was appropriate for adhering 20-30 µm stem cells. F-127 containing SOX9 pDNA, in combination with DOPA-polyethylene-coated gold nanoparticles and dexamethasone loaded in PMcs, promoted cartilage differentiation. Gold nanoparticles complex and dexamethasone (DEX) loaded in PMcs were identified by micro-CT imaging and fluorescence imaging, respectively. By captured in pore generated on/in microspheres, the stem cells were safe and stable for use in delivery, both in vitro and in an animal model. Thus, microsphere pores can safely capture stem cells, and at the same time provide a microenvironment in which the captured stem cells can differentiate into chondrocytes.

Osteoconductive hybrid hyaluronic acid hydrogel patch for effective bone formation.

Bio-inspired adhesive hydrogels have been applied to cell and drug delivery systems to address various tissue defects and disorders. However, adhesive hydrogels functionalized with phenolic moieties often lack osteoconductive capacity and mechanical properties for bone regeneration. In this study, we utilized the versatile chemical interactions of phenolic moieties to overcome such limitations in bone tissue engineering efforts. Highly osteoconductive hybrid hydrogel patches were fabricated by incorporating inorganic minerals, hydroxyapatite (HAP), or whitlockite (WKT), into pyrogallol-conjugated hyaluronic acid (HA-PG). The hybrid HA-PG patches exhibited improved mechanical strength and reinforced structural/physical properties owing to additional intermolecular complexation between oxidized PG moieties and ions released from inorganic particles. The sustained release of bone morphogenetic protein-2 (BMP-2) from hybrid patches was prolonged by combination of the inherent nucleophilic affinity of oxidized PG and electrostatic interactions between inorganic particles and BMP-2. With increased osteoconductivity, hybrid patches with HAP or WKT enhanced the osteogenic differentiation of human stem cells while also promoting new bone formation in a critical-sized calvarial defect. Our study demonstrates a translational potential of phenolic adhesive hydrogels engineered with inorganic minerals for orthopedic applications.

[Factors associated with liver stiffness in chronic liver disease].

BACKGROUND/AIMS: Transient elastography is a new noninvasive tool for measuring liver stiffness that accurately predicts significant fibrosis and cirrhosis. However, several studies have indicated that liver stiffness can be significantly influenced by major changes in aminotransferase in patients with chronic viral hepatitis. The aim of this study was to determine the factors influencing liver stiffness in patients with chronic liver disease. METHODS: We studied 158 patients with chronic liver disease who underwent transient elastography and liver biopsy sampling. Histologic findings on fibrosis and necroinflammatory activity in the biopsy specimens were evaluated according to the Korean Society of Pathologists Scoring System. Routine biochemical tests were performed according to standard methods. RESULTS: Liver stiffness was strongly correlated with liver fibrosis stage (Spearman coefficient=0.636, P<0.001), lobular activity (Spearman coefficient=0.359, P<0.001), and portoperiportal activity grade (Spearman coefficient=0.448, P<0.001). Liver stiffness was significantly associated with serum levels of total bilirubin (P=0.025), direct bilirubin (P=0.049), gamma-glutamyl transpeptidase (P=0.014), platelet count (P=0.004), albumin (P<0.001), and international normalized ratio (P<0.001). Multivariate analysis showed that fibrosis stage (B 3.50, P=0.009) and lobular activity grade (B 3.25, P=0.047) were independently associated with liver stiffness. CONCLUSIONS: Liver stiffness as measured by transient elastography is associated with the grade of necroinflammatory activity and the stage of fibrosis, irrespective of serum ALT levels.

[Comparison of radiofrequency ablation and transarterial chemoembolization for the treatment of a single hepatocellular carcinoma smaller than 4 cm].

BACKGROUND/AIMS: Radiofrequency ablation (RFA) is an established curative therapeutic modality for unresectable hepatocellular carcinoma (HCC), and transarterial chemoembolization (TACE) has been used as a palliative treatment for inoperable HCC. It is still unknown whether RFA and TACE are equally effective for improving the survival of patients with unresectable HCC that is amenable to either treatment. The aim of this retrospective study was to compare the clinical impacts of two treatments, and analyze the prognostic factors for recurrence and survival. METHODS: Ninety-three patients with a single HCC smaller than 4 cm who showed complete responses (complete ablation or complete lipiodol tagging) after treatment with RFA (n=43) or TACE (n=50) between January 2002 and February 2009 were investigated. Univariate and multivariate analyses were performed for 13 potential prognostic factors using the Cox proportional-hazards model. RESULTS: The time-to-recurrence rates at 1, 2, and 3 years after treatment were 32.9%, 44.3%, and 55.4%, respectively, for the RFA group, and 42%, 68.3%, 71.7% for the TACE group. The probability of survival at 1, 2, and 3 years was 97.7%, 77.4%, and 63.1%, respectively, for the RFA group, and 95.9%, 76.1%, and 60.2% for the TACE group. The time-to-recurrence and overall survival rates did not differ significantly between the two treatment groups. A multivariate Cox proportional-hazards model revealed that a tumor size larger than 3 cm and lower serum albumin levels were independent risk factors for recurrence, and that being male, being seropositive for hepatitis B surface antigen, and having a higher serum albumin level were independent favorable prognostic factors for survival. CONCLUSIONS: TACE and RFA exhibited similar therapeutic effects in terms of recurrence and survival for patients with a single HCC smaller than 4 cm, if they could exhibited complete responses.

Coacervate-mediated exogenous growth factor delivery for scarless skin regeneration.

Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-ß3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-ß3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. STATEMENT OF SIGNIFICANCE: This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration.

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L-lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose-derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG-coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52 ± 28.10%) as compared to defect (17.48 ± 11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.

Accelerated Bone Regeneration via Three-Dimensional Cell-Printed Constructs Containing Human Nasal Turbinate-Derived Stem Cells as a Clinically Applicable Therapy.

Stem cell transplantation is a promising therapeutic strategy that includes both cell therapy and tissue engineering for the treatment of many regenerative diseases; however, the efficacy and safety of stem cell therapy depend on the cell type used in therapeutic and translational applications. In this study, we validated the hypothesis that human nasal turbinate-derived mesenchymal stem cells (hTMSCs) are a potential therapeutic source of adult stem cells for clinical use in bone tissue engineering using three-dimensional (3D) cell-printing technology. hTMSCs were cultured and evaluated for clinical use according to their cell growth, cell size, and preclinical safety and were then incorporated into a multicompositional 3D bioprinting system and investigated for bone tissue regeneration in vitro and in vivo. Finally, hTMSCs were compared with human bone marrow-derived MSCs (hBMSCs), which are the most common stem cell type used in regenerative medicine. hTMSCs from three different donors showed greater and faster cell growth than hBMSCs from two different donors when cultured. The hTMSCs were smaller in size than the hBMSCs. Furthermore, the hTMSCs did not exhibit safety issues in immunodeficient mice. hTMSCs in 3D-printed constructs (3D-hTMSC) showed much greater viability, growth, and osteogenic differentiation potential in vitro than hBMSCs in 3D-printed constructs (3D-hBMSC). Likewise, 3D-hTMSC showed better cell survival and alkaline phosphatase activity and greater osteogenic protein expression than 3D-hBMSC upon subcutaneous implantation into the dorsal region of nude mice. Notably, in an orthotopic model involving implantation into a tibial defect in rats, implantation of 3D-hTMSC led to greater bone matrix formation and enhanced bone healing to a greater degree than implantation of 3D-hBMSC. The clinically reliable evidence provided by these results is underlined by the potential for rapid tissue regeneration and ambulation in bone fracture patients implanted with 3D-hTMSC.

Differentiated osteoblasts derived decellularized extracellular matrix to promote osteogenic differentiation.

BACKGROUND: The extracellular matrix (ECM) can directly or indirectly influence on regulation of cell functions such as cell adhesion, migration, proliferation and differentiation. The cell derived ECM (CD-ECM) is a useful in vitro model for studying the comprehensive functions of CD-ECM because it maintains a native-like structure and composition. In this study, the CD-ECM is obtained and a test is carried out to determine the effectiveness of several combinations of decellularized methods. These methods were used to regulate the optimal ECM compositions to be induced by osteogenic differentiation using primary isolated osteoblasts. RESULT: We investigated the effect of osteoblasts re-seeded onto normal osteoblast ECM under the growth medium (GM-ECM) and the osteogenic differentiation medium (OD-ECM). The osteoblasts were then cultured statically for 1, 2, and 4 weeks in a growth medium or differentiation medium. Before osteoblast culture, we performed immunostaining with filamentous actin and nuclei, and then performed DNA quantification. After each culture period, the osteogenic differentiation of the osteoblasts re-seeded on the OD-ECMs was enhanced osteogenic differentiation which confirmed by alkaline phosphatase staining and quantification, Alizarin Red S staining and quantification, and von Kossa staining. The OD-ECM-4 W group showed more effective osteogenic differentiation than GM-ECM and OD-ECM-2 W. CONCLUSIONS: The OD-ECM-4 W has a better capacity in a microenvironment that supports osteogenic differentiation on the GM-ECM and OD-ECM-2 W. The ECM substrate has a wide range of applications as cell culture system or direct differentiation of stem cell and excellent potential as cell-based tissue repair in orthopedic tissue engineering.

Topographically Defined, Biodegradable Nanopatterned Patches to Regulate Cell Fate and Acceleration of Bone Regeneration.

If only allowed to proceed naturally, the bone-healing process can take several weeks, months, or even years depending on the injury size. In terms of bone-healing speed, many studies have been conducted investigating the deliverance of various growth factors of implantable biomaterials to shorten the time for bone regeneration. However, there may be side effects such as nerve pain, infection, or ectopic bone formation. As an alternative method, we focused on biophysical guidance, which provided similar topographical cues to the cellular environment to recruit host cells for bone defect healing. In this study, we hypothesized that aligned nanotopographical features have enhanced osteoblast recruitment, migration, and differentiation without external stimuli. We designed and fabricated a biodegradable poly(lactic- co-glycolic acid) nanopatterned patch using simple solvent casting and capillary force lithography. We confirmed that a biodegradable nanopatterned patch (BNP) accelerated the migration of osteoblasts according to the orientation of the patterned direction. These highly aligned osteoblasts may contribute to in vitro osteogenic differentiation, such as alkaline phosphate activity, mineralization, and calcium deposition, compared to the biodegradable flat patch (BFP). To demonstrate bone defect healing by BNP guidance in vivo, we implanted either whole or bridge BNP on the critical size defect of mouse calvarial ( ø 4 mm) or tibia bone (3 × 7 mm2). Only the BNP-treated group showed faster new bone formation and compact bone regeneration at the calvarial or tibia bone defect area compared to BFP at 4 or 8 weeks. Bridge BNP guided, in particular, the regeneration of new bone formation along the parallel direction of nanopatterned substrates. Here, we show that a BNP with biophysical guidance should be suitable for use in bone tissue regeneration through accelerated migration of the intact host cell.

Biodegradable Nerve Guidance Conduit with Microporous and Micropatterned Poly(lactic-co-glycolic acid)-Accelerated Sciatic Nerve Regeneration.

An innovative technique combining capillary force lithography and phase separation method in one step is applied to fabricate artificial nerve guidance conduit (NGC) for peripheral nerve regeneration. Biodegradable porous, patterned NGC (PP-NGC) using poly(lactic-co-glycolic acid) is fabricated. It has micro-grooves and microporosity on the inner surface to promote axonal outgrowth and to enhance permeability for nutrient exchange. In this study, it is confirmed that the inner surface of micro-grooves can modulate neurite orientation and length of mouse neural stem cell compared to porous flat NGC (PF-NGC) in vitro. Coating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitates the hydrophilic inner surface of PF- and PP-NGCs via bioinspired catechol chemistry. For in vivo study, PF-NGC and PP-NGC coated with or without DOPA are implanted in the 10 mm sciatic nerve defect margins between proximal and distal nerves in rats. Especially, PP-NGC coated with DOPA shows higher sciatic function index score, onset-to-peak amplitude, and muscle fiber diameter compared to other groups. The proposed hybrid-structured NGC not only can serve as a design for functional NGC without growth factor but also can be used in clinical application for peripheral nerve regeneration.