We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.
We have observed the efficiency of antibiotic-releasing polylactide-co-glycolide (PLGA) 80/20 in preventing Staphylococcus epidermidis attachment and biofilm formation in vitro. The aim of the present study was to evaluate the effect of self-reinforced (SR) implants with enhanced antibiotic release on bacterial attachment and biofilm formation rates, and also on growth inhibition of Staphylococcus epidermidis. Cylindrical SR-PLGA+AB specimens (length 30 mm, diameter 3 mm) were examined by scanning electron microscopy (SEM) for attachment of S. epidermidis ATCC 35989 on biomaterial surface and formation of biofilm, after incubating with bacterial suspension of ca. 10 cfu/mL for 1, 3, 7, 14 and 21 days. SR-PLGA and SR-PLGA+AB implants were tested on agar plates by measuring the inhibition distance around implants. On the surface of SR-PLGA+AB, at days 1, 3, 7, 14 and 21, the percentage of areas with not a single bacteria attached, was 88.6%, 71.1%, 73.7%, 73.7%, and 68.4%, respectively. On the areas where bacteria were detected, the number of bacterial cells remained low during whole study period, and no significant increase by time was seen. There was no biofilm observed on 97-99% of the examined areas during the whole study period on SR-PLGA+AB. In agar plates, the SR-PLGA+AB showed inhibition of bacterial growth, with (mean) 53.2 mm diameter of inhibition area with peeled implants and 50.5 mm with non-peeled implants. There was no inhibition seen around implants without ciprofloxacin. Bioabsorbable ciprofloxacin-releasing self-reinforced PLGA (SR-PLGA+AB) was superior to plain SR-PLGA in preventing bacterial attachment, biofilm formation, and also the growth of Staphylococcus epidermidis.
To develop a successful bone fixation device that can also release therapeutic agents such as antibiotics one has to consider mechanical, drug release, and biocompatibility properties. We have used bioabsorbable polymers (PLGA 80/20 or PLDLA 70/30) as the matrix and ciprofloxacin (CF) as antibiotic to develop such an implant. Initial shear strengths of the studied ciprofloxacinreleasing screws were 152 MPa (P/L/DL)LA) and 172 MPa (PLGA). Studied screws retained their mechanical properties for least 12 weeks (P(L/DL)LA) and 9 weeks (PLGA) in vitro at the level that ensures their fixation properties. Pull-out tests indicated that the early version of screws have lower values as compared to controls. CF was found to be released after 44 weeks (P/L/DL)LA) and 23 weeks (PLGA) in vitro. It remained in the range of 0.06 – 8.7 µg/ml/day (for P(L/DL)LA) and 0.6 - 11.6 µg/ml/day (for PLGA) after the jump start. Release CF was demonstrated to significantly inhibit S. epidermides growth, attachment and biofilm formation different than controls. Histology showed no difference from plain polymer screws, except for increased giant cells at the implantation site. Accordingly, SR-P(L/DL)LA and SR-PLGA MF implants were considered appropriate to proceed to pilot clinical application.
Transplantation of autologous fat as pedicle or transposition flaps has been a classical method in plastic surgery for tissue reconstruction. The injection of fat for soft tissue reconstruction is also an old innovation. This approach has some significant drawbacks such as resorption of the fat transplant. To regenerate additional and self-regenerating adipose tissue for reconstructive purposes, a thorough understanding of adipose tissue (mesodermal stem cells, adipoblasts, pre-adipocytes, mature, lipid-synthesizing, and lipid-storing white or brown adipocytes) on cellular and molecular levels is required. Several transcription factors that play a central role in the control of adipogenesis have been identified. Among these are the CCAAT/enhancer binding protein gene family and peroxisome proliferator-activated receptor-gamma. Hormones and growth factors, such as insulin and insulin-like growth factor (IGF), transfer external signals to differentiating adipocytes. In an attempt to improve the quality of tissue-engineered fat by culture-expanded adipocytes, various pre-adipocyte and stem cell culture conditions and expansion methods have been developed. In the presence of fetal calf serum, spontaneous differentiation of pre-adipocytes into fat cell clusters occurs to some degree. This in vitro differentiation can be enhanced by addition of inducing agents such as dexamethasone, isobutylmethylxantine, and insulin into the culture medium. Recent work has shown the multipotency of pre-adipocytes, which are fibroblast-like precursors of adipocytes. With use of specific culture conditions, human adipose tissue-derived stem cells can be induced to express markers of adipocyte, osteoblast, and myocyte cell lineages. The multipotent characteristics of adipose tissue-derived stem cells, as well as their abundance and accessibility in the human body, make them a potential cell source for tissue engineering applications.
