A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

Substrate-induced phenotypical change of monocytes/macrophages into myofibroblast-like cells: a new insight into the mechanism of in-stent restenosis.

Stented coronary angioplasty is the procedure of choice to re-establish patency in obstructed coronary arteries. However, the stent implantation procedure often leads to in-stent restenosis, a process that is characterized by stent strut colonization by macrophages and smooth muscle cells and by neointima formation. The present in vitro study investigates the effect of stent materials on the phenotypical features of monocyte/macrophages. Human peripheral blood monocytes from healthy donors (n = 7) were cultured up to 7 days on substrates mimicking: (i) the stent surface (i.e., electropolished stainless steel), (ii) the de-endothelialized vessel wall (collagen-based extracellular matrix gel), and (iii) thrombus (i.e., fibrin gel). The cells were analyzed by immunocytochemistry for their ability to express alpha-actin, a typical myofibroblast marker, by ELISA to determine PDGF-BB and TGF-beta1 secretion and by PCR to evaluate hyaluronan synthase 1, 2, and 3 genes expression. Data were statistically analyzed by ANOVA (Dunnett's test) and data considered significantly different at p

The internervous safe zone for incision of the capsule of the hip. A cadaver study.

In order to determine the potential for an internervous safe zone, 20 hips from human cadavers were dissected to map out the precise pattern of innervation of the hip capsule. The results were illustrated in the form of a clock face. The reference point for measurement was the inferior acetabular notch, representing six o'clock. Capsular branches from between five and seven nerves contributed to each hip joint, and were found to innervate the capsule in a relatively constant pattern. An internervous safe zone was identified anterosuperiorly in an arc of 45 degrees between the positions of one o'clock and half past two. Our study shows that there is an internervous zone that could be safely used in a capsule-retaining anterior, anterolateral or lateral approach to the hip, or during portal placement in hip arthroscopy.