Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.     

Evaluation of antibiotic-loaded collagen-hyaluronic acid matrix as a skin substitute

The 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride-crosslinked collagen-hyaluronic acid (HA) matrices containing tobramycin or ciprofloxacin as an antibiotic agent were fabricated for the control of wound contamination and characterized with respect to morphology, mechanical strength, in vitro release, antibacterial activity and cytotoxicity. For the tobramycin loaded matrix, the antibacterial capacity increased with the drug loading. Tobramycin and ciprofloxacin loaded matrices maintained their antibacterial effects for over 96 and 48 h, respectively. However, cell viability testing revealed that 0.4 mg/ml of ciprofloxacin has a cytotoxic effect on fetal human dermal fibroblasts. The effects of the bilayered collagen-HA matrices containing tobramycin and growth factors were also evaluated using an in vivo full thickness dermal defect model. Though the tobramycin incorporated collagen-HA matrix had no significant effect on wound healing compared with the control, the tobramycin incorporated matrix containing basic fibroblast growth factor or platelet-derived growth factor significantly enhanced wound healing. This study demonstrates the potential efficacy of crosslinked collagen-HA matrix containing antibiotics and growth factors for defective skin tissue replacement and infection prevention.     

A polyethylene glycol grafted bi-layered polyurethane scaffold: preliminary study of a new candidate prosthesis for repair of a partial tracheal defect

The purpose of this study was to develop an artificial prosthesis for use in the reconstruction of a tracheal defect due to trauma, malignancy, stenosis, or other causes. Bi-layered porous-dense film polyurethane (PU) was manufactured for the main framework. Polyethylene glycol (PEG) was grafted onto the inner surface of the PU scaffold to act as a surfactant. The scaffold was transplanted into three beagles. An endoscopic examination was performed for the evaluation of the formation granulation tissue, to evaluate the status of the respiratory mucosa and the amount of crust at 1, 4, 8, and 12 weeks after surgery. A histological examination was also performed at 4, 8, and 12 weeks after surgery. All three beagles studied survived to the expected date. The endoscopic examination showed formation of granulation tissue; the crust was not very severe and the circular tracheal framework was well preserved. The histological examination showed that a large amount of fibrous tissue had grown through the pores of the porous scaffold. Pseudostratified columnar ciliated mucosa was also noted on the surface of scaffold, as visualized by scanning electron microscopy. The use of a bi-layered polyethylene grafted polyurethane scaffold is a good candidate prosthesis for tracheal reconstruction.     

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow‐derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) undergo senescence‐related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5‐azacytidine was not associated with the phenomenon, but the serum‐starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM‐MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle‐shape morphology, increased in proliferation rate by two‐fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.     

A phase II study of combination chemotherapy with gemcitabine, 5-fluorouracil, and cisplatin for advanced pancreatic cancer]

BACKGROUND/AIMS: Gemcitabine has been the standard regimen for advanced pancreatic cancer, but the effect on the response rate and survival is still disappointing, leading to many trials of combination chemotherapy. 5-FU and cisplatin were combined with gemcitabine in this trial, as they are synergistic with gemcitabine and each other as well. This study was aimed to assess the effectiveness and safety of combination chemotherapy with gemcitabine, 5-FU, and cisplatin for advanced pancreatic cancer. METHODS: Patients with advanced pancreatic cancer were entered into this study. Gemcitabine at a dose of 800 mg/m2 on day 1 and 8, 5-FU 1,000 mg/m2/day from day 1 to 3 for 72 hours, and cisplatin 60 mg/m2 on day 2, 24 hours after the start of gemcitabine were administered every 3 weeks. RESULTS: From December 2001 to January 2004, twenty patients were enrolled in this study. Among 17 of these patients assessable, 3 patients had a partial remission with the response rate of 23.6% (95% confidence interval, 6.2-41.0%). The median time to disease progression was 230 days and median duration of survival was 322 days. Among total of 91 cycles, leukopenia, neutropenia, and thrombocytopenia of grade 3 or 4 occurred in 12 cycles (13.2%), 12 cycles (13.2%), and 23 cycles (24.4%), respectively. Grade 3 or 4 mucositis developed at 2 cycles (2.2%), and nausea and vomiting were encountered in 3 cycles (3.3%). CONCLUSIONS: Combination chemotherapy with gemcitabine, 5-FU, and cisplatin for advanced pancreatic cancer is active and well-tolerated, warranting a phase III study.     

Construction of functional soft tissues from premodulated smooth muscle cells using a bioreactor system

Artificial smooth muscle tissues should be constructed with well-differentiated and aligned smooth muscle cells (SMCs) for proper functioning. In a previous study, we produced cell/scaffold hybrids composed of consistently aligned SMCs in a contractile state using cyclic mechanical strain. In this study, the preconditioned hybrids were organized as functional smooth muscle constructs, which had a high cellular density, using a bioreactor system. We determined that the alignment and contractile phenotype of the initially generated SMCs would be retained after a 7-day culture period in a bioreactor. Mechanical properties of the smooth muscle constructs were measured and compared with those of native smooth muscle tissues and acellular scaffolds. The constructs had a denser cell concentration than the preconditioned hybrids, although they were not fully filled with cells. The premodulated cell alignment and contractile phenotype were retained after culture in a bioreactor. The 7-day-cultured constructs had similar allowed stress levels to native tissues while their stiffness was much lower, suggesting that they had malleable and durable characteristics. These results suggest that functional smooth muscle tissues with mechanical stability can be produced using premodulated SMCs and a bioreactor system.     

Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres

The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPAR(gamma) and C/EBP(alpha) were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.     

The prevention of vocal fold scarring using autologous adipose tissue-derived stromal cells

Prevention and treatment of vocal fold scarring and atrophy remain challenging. The aim of this study was to treat injured vocal folds using autologous adipose tissue-derived stromal cells (ADSCs) and evaluate the ability to prevent vocal fold scarring and atrophy by ADSCs in a canine animal model. Ten adult dogs were used for this experiment. ADSCs from the adipose tissue from the inguinal area were isolated and cultured in all dogs. Immediately after being mixed with atelocollagen, the ADSCs (1-3 x 10(6)) were injected into the right vocal fold of each animal, using a syringe with a 23-gauge needle. As a control, atelocollagen was injected into the left vocal fold of the same dog. The effects of the prevention of vocal fold scarring and atrophy were measured by morphological and histological assessment. At 8 weeks, there was a difference in granuloma and atrophic changes between the ADSC-injected and control sides in the majority of the dogs. This difference continued to be present at the 24 weeks' follow-up. On histopathologic examination, a large number of cells labeled with a fluorochrome were observed in ADSC-injected vocal folds 8 weeks after the initial treatment. This study demonstrates the multipotential ability of ADSCs in the regeneration of injured vocal folds. Injecting ADSCs into a damaged vocal fold appears to be useful in preventing vocal fold scarring and atrophy 24 weeks after initial damage.     

Inhibin B as a screening tool for early detection and treatment monitoring of central precocious puberty

Abstract

Anti-Müllerian hormone (AMH) and inhibin B are considered possible biomarkers of central precocious puberty (CPP). The aim of this study was to evaluate serum levels of AMH and inhibin B, to investigate their regulatory patterns, and to study their clinical significance in girls with CPP. In total, 48 girls with CPP and 35 age-matched prepubertal control girls were enrolled in the study. AMH and inhibin B levels were determined in the CPP and control groups. In the patient group, AMH and inhibin B levels were evaluated during 1?year of gonadotropin releasing hormone analog (GnRHa) treatment. The mean inhibin B level in the CPP group was significantly higher than that in the control. AMH levels were not different between the two groups. After GnRHa treatment. AMH and inhibin B levels decreased significantly. Based on the ROC analysis, the cutoff value for inhibin B to determine CPP was 19.59?pg/mL, with 83.3% sensitivity and 82.9% specificity, and the area under the curve was 0. 852. Inhibin B was useful for determining CPP and the therapeutic effects of GnRHa treatment in girls with CPP. AMH interacted, in part, with the hypothalamo-pituitary gonadal axis, but its clinical implications in CPP should be further investigated.     

Serum Makorin ring finger protein 3 values for predicting Central precocious puberty in girls

This study evaluated the serum level of MKRN3 and investigated its diagnostic usefulness in girls with central precocious puberty (CPP). In total, 41 girls with CPP and 35 age-matched normal control girls were enrolled. Serum values of MKRN3 were measured in both groups. Gonadotropin and estradiol concentrations were evaluated after 6 and 12?months of GnRH agonist (GnRHa) treatment in CPP patients. The MKRN3 concentrations were much lower in the patient group than in the control group (p?=?.005). Over 1 year of GnRHa treatment in patients, the gonadotropin concentrations were significantly decreased (p?<?.05), while the MKRN3 concentrations were unchanged (p?>?.05). MKRN3 levels were inversely correlated to standard deviation (SD) in height (r?=??0.46, p?=?.000), SD in weight (r?=??0.32, p?=?.005), Tanner stage (r?=??0.41, p?=?.000), and bone age (r?=??0.46, p?=?.000). Based on ROC analysis, the area under curve was 0.758 for MKRN3, with 82.9% sensitivity and 68.5% specificity. The measurement of serum MKRN3 level may provide some help for CPP prediction, but relatively various values need further validation