Development and intra-renal delivery of renal progenitor organoids for effective integration in vivo.

Renal progenitor organoids have been proposed as a source of tissue for kidney regeneration; however, their clinical translatability has not been demonstrated due to an inability to mass-produce comprehensive renal progenitor organoids and the lack of an effective intra-renal delivery platform that facilitates rapid integration into functionally meaningful sites. This study addresses these shortcomings. Human-induced pluripotent stem cells were differentiated into renal progenitor cells using an established protocol and aggregated using a novel assembly method to produce high yields of organoids. Organoids were encapsulated in collagen-based scaffolds for in vitro study and in vivo implantation into mouse renal cortex. In vitro, the organoids demonstrated sustained cell viability and renal structure maturation over time. In vivo delivered organoids showed rapid integration into host renal parenchyma while showing tubular and glomerular-like structure development and maturity markers. This proof-of-concept study presents many promising results, providing a system of renal organoid formation and delivery that may support the development of clinically translatable therapies and the advancement of in vitro renal organoid studies.

Enhancing Craniofacial Bone Reconstruction with Clinically Applicable 3D Bioprinted Constructs.

Medical imaging and 3D bioprinting can be used to create patient-specific bone scaffolds with complex shapes and controlled inner architectures. This study investigated the effectiveness of a biomimetic approach to scaffold design by employing geometric control. The biomimetic scaffold with a dense external layer showed improved bone regeneration compared to the control scaffold. New bone filled the defected region in the biomimetic scaffolds, while the control scaffolds only presented new bone at the boundary. Histological examination also shows effective bone regeneration in the biomimetic scaffolds, while fibrotic tissue ingrowth is observed in the control scaffolds. These findings suggest that the biomimetic bone scaffold, designed to minimize competition for fibrotic tissue formation in the bony defect, can enhance bone regeneration. This study underscores the notion that patient-specific anatomy can be accurately translated into a 3D bioprinting strategy through medical imaging, leading to the fabrication of constructs with significant clinical relevance.

Pelvic floor muscle function recovery using biofabricated tissue constructs with neuromuscular junctions.

Damages in pelvic floor muscles often cause dysfunction of the entire pelvic urogenital system, which is clinically challenging. A bioengineered skeletal muscle construct that mimics structural and functional characteristics of native skeletal muscle could provide a therapeutic option to restore normal muscle function. However, most of the current bioengineered muscle constructs are unable to provide timely innervation necessary for successful grafting and functional recovery. We previously have demonstrated that post-synaptic acetylcholine receptors (AChR) clusters can be pre-formed on cultured skeletal muscle myofibers with agrin treatment and suggested that implantation of AChR clusters containing myofibers could accelerate innervation and recovery of muscle function. In this study, we develop a 3-dimensional (3D) bioprinted human skeletal muscle construct, consisting of multi-layers bundles with aligned and AChR clusters pre-formed human myofibers, and investigate the effect of pre-formed AChR clusters in bioprinted skeletal muscle constructs and innervation efficiency in vivo. Agrin treatment successfully pre-formed functional AChR clusters on the bioprinted muscle constructs in vitro that increased neuromuscular junction (NMJ) formation in vivo in a transposed nerve implantation model in rats. In a rat model of pelvic floor muscle injury, implantation of skeletal muscle constructs containing the pre-formed AChR clusters resulted in functional muscle reconstruction with accelerated construct innervation. This approach may provide a therapeutic solution to the many challenges associated with pelvic floor reconstruction resulting from the lack of suitable bioengineered tissue for efficient innervation and muscle function restoration.

Neural cell integration into 3D bioprinted skeletal muscle constructs accelerates restoration of muscle function.

A bioengineered skeletal muscle construct that mimics structural and functional characteristics of native skeletal muscle is a promising therapeutic option to treat extensive muscle defect injuries. We previously showed that bioprinted human skeletal muscle constructs were able to form multi-layered bundles with aligned myofibers. In this study, we investigate the effects of neural cell integration into the bioprinted skeletal muscle construct to accelerate functional muscle regeneration in vivo. Neural input into this bioprinted skeletal muscle construct shows the improvement of myofiber formation, long-term survival, and neuromuscular junction formation in vitro. More importantly, the bioprinted constructs with neural cell integration facilitate rapid innervation and mature into organized muscle tissue that restores normal muscle weight and function in a rodent model of muscle defect injury. These results suggest that the 3D bioprinted human neural-skeletal muscle constructs can be rapidly integrated with the host neural network, resulting in accelerated muscle function restoration.

A novel decellularized skeletal muscle-derived ECM scaffolding system for in situ muscle regeneration.

The cell-based tissue engineering strategies have gained attention in restoring normal tissue function after skeletal muscle injuries; however, these approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation. In order to avoid this limitation, we developed a novel cell-free muscle-specific scaffolding system that consisted of a skeletal muscle-derived decellularized extracellular matrix (dECM) and a myogenic factor, insulin growth factor-1 (IGF-1). Rheological, morphological, and biological properties of this muscle-specific scaffold (IGF-1/dECM) as well as collagen and dECM scaffolds were examined. The cell viability in all scaffolds had over 90% at 1, 3, and 7 days in culture. The cell proliferation in the IGF-1/dECM was significantly increased when compared with other groups. More importantly, the IGF-1/dECM strongly supported the myogenic differentiation in the scaffold as confirmed by myosin heavy chain (MHC) immunofluorescence. We also investigated the feasibility in a rabbit tibialis anterior (TA) muscle defect model. The IGF-1/dECM had a significantly greater number of myofibers when compared to both collagen and dECM groups at 1 and 2 months after implantation. We demonstrated that this novel muscle-specific scaffolding system could effectively promote the muscle tissue regeneration in situ.

Whole kidney engineering for clinical translation.

PURPOSE OF REVIEW: Renal transplantation is currently the only definitive treatment for end-stage renal disease; however, this treatment is severely limited by the shortage of implantable kidneys. To address this shortcoming, development of an engineered, transplantable kidney has been proposed. Although current advances in engineering kidneys based on decellularization and recellularization techniques have offered great promises for the generation of functional kidney constructs, most studies have been conducted using rodent kidney constructs and short-term in-vivo evaluation. Toward clinical translations of this technique, several limitations need to be addressed. RECENT FINDINGS: Human-sized renal scaffolds are desirable for clinical application, and the fabrication is currently feasible using native porcine and discarded human kidneys. Current progress in stem cell biology and cell culture methods have demonstrated feasibility of the use of embryonic stem cells, induced pluripotent stem cells, and primary renal cells as clinically relevant cell sources for the recellularization of renal scaffolds. Finally, approaches to long-term implantation of engineered kidneys are under investigation using antithrombogenic strategies such as functional reendothelialization of acellular kidney matrices. SUMMARY: In the field of bioengineering, whole kidneys have taken a number of important initial steps toward clinical translations, but many challenges must be addressed to achieve a successful treatment for the patient with end-stage renal disease.

Diet control to achieve euglycemia induces significant loss of heart and liver weight via increased autophagy compared with ad libitum diet in diabetic rats.

Intensive glucose control increases the all-cause mortality in type 2 diabetes mellitus (T2DM); however, the underlying mechanisms remain unclear. We hypothesized that strict diet control to achieve euglycemia in diabetes damages major organs, increasing the mortality risk. To evaluate effects on major organs when euglycemia is obtained by diet control, we generated a model of end-stage T2DM in 13-week-old Sprague-Dawley rats by subtotal pancreatectomy, followed by ad libitum feeding for 5 weeks. We divided these rats into two groups and for the subsequent 6 weeks provided ad libitum feeding to half (AL, n=12) and a calorie-controlled diet to the other half (R, n=12). To avoid hypoglycemia, the degree of calorie restriction in the R group was isocaloric (g per kg body weight per day) compared with a sham-operated control group (C, n=12). During the 6-week diet control period, AL rats ate three times more than rats in the C or R groups, developing hyperglycemia with renal hyperplasia. R group achieved euglycemia but lost overall body weight significantly compared with the C or AL group (49 or 22%, respectively), heart weight (39 or 23%, respectively) and liver weight (50 or 46%, respectively). Autophagy levels in the heart and liver were the highest in the R group (P<0.01), which also had the lowest pAkt/Akt levels among the groups (P<0.05 in the heart; P<0.01 in the liver). In conclusion, glycemic control achieved by diet control can prevent hyperglycemia-induced renal hyperplasia in diabetes but may be deleterious even at isocaloric rate when insulin is deficient because of significant loss of heart and liver mass via increased autophagy.

Improvement of ß-cell function after achievement of optimal glycaemic control via long-term continuous subcutaneous insulin infusion therapy in non-newly diagnosed type 2 diabetic patients with suboptimal glycaemic control.

BACKGROUND: Achieving euglycaemia by continuous subcutaneous insulin infusion (CSII) therapy alone has been shown to restore ß-cell function in patients with newly diagnosed type 2 diabetes. However, the efficacy has not been evaluated in patients with non-newly diagnosed type 2 diabetes and suboptimal glycaemic control. METHODS: Of the 1220 patients with type 2 diabetes who began CSII therapy from March 2000 to March 2007, we retrospectively selected patients using the following inclusion criteria: glycosylated haemoglobin (HbA1c ) ≥ 7.0%, diabetes duration ≥ 1 year before CSII therapy, and duration of CSII therapy ≥ 6 months. We evaluated sequential changes in HbA1c and serum C-peptide levels measured at a 6- to 12-month intervals during CSII therapy. RESULTS: In the 521 subjects included in this study [median diabetes duration 10 years; interquartile range (IQR) 6.0-17.0; CSII therapy ≤ 30 months], median HbA1c decreased from 8.7% (IQR 7.7-10.0) at baseline to 6.3% (IQR 5.9-6.9) after 6 months of CSII therapy (p < 0.0001). During the subsequent 24 months, median HbA1c levels were maintained between 6.3% and 6.5% (p < 0.0001 for all time points vs baseline). At 12 months after CSII therapy, median C-peptide levels began to increase compared with baseline (fasting level 23% increase, p < 0.0001; 2-h postprandial level 26% increase, p = 0.022), and the increase was maintained at 30 months (fasting level 39%; 2-h postprandial level 53%; p < 0.0001 for all vs baseline). CONCLUSIONS: ß-Cell function was significantly improved in patients with non-newly diagnosed and suboptimally controlled type 2 diabetes after achieving and maintaining optimal glycaemic control with long-term CSII therapy alone.

Acorus gramineus inhibits microglia mediated neuroinflammation and prevents neurotoxicity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorus gramineus Solander (Acoraceae, AG), is a widely distributed plant in Asian countries. Rhizome part of this plant has long been used as a traditional medicine for treating various symptoms including central nervous system (CNS) disorders. AIM OF STUDY: The anti-neuroinflammatory effect of AG aqueous extract was investigated using in vitro cellular and in vivo Parkinson's disease (PD) mouse model. MATERIALS AND METHODS: Lipopolysaccharide (LPS) is used to stimulate BV-2 microglial cells in vitro and the changes in neuroinflammatory expressional levels were measured using ELISA, Western blotting, RT-PCR and immunofluorescence techniques. In in vivo experiments, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD was developed followed by immunohistochemical analysis of specific brain tissues. RESULTS: LPS-stimulation to BV-2 cells increased the production of nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß. Pretreatment with AG extract inhibited the increased levels of NO and pro-inflammatory cytokines in LPS-stimulated BV-2 cells. Mechanistic study revealed that AG acts via the regulation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPKs) and TRIF-dependent signaling pathways. Further, AG protected MPTP-induced neuronal cell death and inhibited neuroinflammation in vivo. CONCLUSION: Our results indicated that AG extract exerted anti-neuroinflammatory effects against activated microglia mediated insults through multiple signaling pathways and prevented in vivo neuronal cell death in mouse model of PD substantiating the traditional claims for its use in CNS disorders.

De novo generation of short antimicrobial peptides with simple amino acid composition.

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L5K2W2), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L4K5W2) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.

Venous hemodynamic changes in the surgical treatment of primary varicose vein of the lower limbs.

Venous hemodynamic changes after the surgery of primary varicose veins were evaluated. (Materials and methods) We retrospectively analyzed 1,211 patients (1,407 limbs) who underwent surgery for primary varicose veins from 1994 to 2002. The venous hemodynamics were evaluated using air- plethysmography (APG) preoperatively and one month postoperatively in the viewpoints of ambulatory venous pressure (AVP), venous volume (VV), venous filling index (VFI), and ejection fraction (EF). (Results) The surgical modalities included 958 cases of greater saphenous vein high ligation (GSV HL) and stripping with varicosectomy (VS), 222 cases of short saphenous vein (SSV) HL and VS, 143 cases of external banding valvuloplasty of GSV and VS, and 44 cases using VNUS and VS. The reduction rate of VV was 20.9 +/- 14.1% in the GSV stripping group, 12.0 +/- 14.7% in the GSV valvuloplasty group, 18.3 +/- 16.1% in the VNUS group, and 20.6 +/- 15.9% in the SSV group. The reduction rate of VFI was 63.6 +/- 20.7% in the GSV stripping group, 38.8 +/- 40.9% in the GSV valvuloplasty group, 60.1 +/- 23.9% in the VNUS group, and 37.6 +/- 30.2% in the SSV group. The increasing rate of EF was 25.0 +/- 28.2% in the GSV stripping group, 21.0 +/- 30.0% in the GSV valvuloplasty group, 29.4 +/- 31.9% in the VNUS group, and 30.0 +/- 36.5% in the SSV group. The reduction rate of AVP was 25.4 +/- 32.2% in the GSV stripping group, -6.1 +/- 58.1% in the GSV valvuloplasty group, 28.4 +/- 38.5% in the VNUS group, and 14.1 +/- 49.0% in the SSV group. All of the patients showed improvements in venous hemodynamics by showing a decrease in VV, VFI, AVP, and an increase in EF. However, there was no difference in the change of venous hemodynamics according to the type of surgery.