Human cell-conditioned media produced under embryonic-like conditions result in improved healing time after laser resurfacing.

BACKGROUND: Laser resurfacing procedures are continuing to grow in popularity as patients select less invasive procedures for rejuvenation of photo-damaged and aging skin. However, although physicians have begun exploring options to aid in postlaser healing, currently available treatments have little clinical evidence to support their use for wounded skin. METHODS: When grown under conditions of very low oxygen and suspension, a simulation of the embryonic environment, neonatal cells have been found to produce proteins and growth factors in types and quantities similar to those of fetal cells. The human cell-conditioned media (hCCM) produced by the cells was extracted and formulated into a gel to evaluate its efficacy in the healing of postlaser wounds. RESULTS: A split-face clinical evaluation of the material was performed, with 42 subjects undergoing combination ablative and nonablative laser procedures. Three concentrations of the hCCM were tested (× 0.1, × 1.0, × 10.0), and a dose-response trend was seen in the blinded physician evaluation, particularly in the assessment of crusting. In addition, transepidermal water loss readings showed a significant difference (p ≤ 0.05), indicating a more rapid return to normal skin barrier function with the active treatment. Histopathologic evaluation of subject biopsies showed reduced inflammation and a more normal epidermal appearance in the active treatment sites. CONCLUSIONS: The results of this clinical evaluation support the use of the soluble hCCM produced under embryonic-like conditions to accelerate wound healing after laser resurfacing procedures. The utility of the × 10 concentration appears to promote more rapid, scarless wound healing after resurfacing procedures and more normal skin recovery.

Acute spinal cord injury: pharmacotherapy and drug development perspectives.

There are no drugs specifically approved for spinal cord injury that directly address the underlying damage to neural tissue. Immediate treatment with methylprednisolone sodium succinate has been shown to improve outcome from injury in a series of preclinical and clinical studies, and it is now widely used, though the benefits appear to be modest. A variety of other approaches to protecting the injured spinal cord from secondary pathological processes have been examined experimentally, including antioxidants, membrane stabilizers, glutamate antagonists, anti-inflammatories, caspase inhibitors, calpain inhibitors and other compounds of uncertain mechanism. All of these approaches have been supported by positive animal studies but their efficacy relative to the widely used methylprednisolone has not been established.

Recruitment of calcium from intracellular stores does not occur during the expression of large spontaneous calcium oscillations in GH(3) cells and lactotropic cells in primary culture.

We used simultaneous electrophysiological and intracellular calcium microfluorometry recordings to directly test for the presence of a calcium-induced calcium release mechanism in individual GH(3) cells and cells of a lactotrope-enriched primary culture. In voltage-pulse experiments, extending the duration of a depolarizing voltage-pulse increased intracellular calcium concentration ([Ca(2+)](i)), but we did not observe any evidence for recruitment of intracellular calcium stores. Furthermore, depletion of intracellular calcium stores with thapsigargin or caffeine did not change the calculated calcium buffer capacity of the cells. In current-clamp experiments, we observed elevations in [Ca(2+)](i) in response to spontaneous action potentials. These [Ca(2+)](i) responses were not inhibited by thapsigargin or caffeine. We did find a significant correlation between the magnitude of spontaneous [Ca(2+)](i) increases and action potential duration. We conclude that intracellular calcium stores are not released during the spontaneous [Ca(2+)](i) oscillations observed in these cells, and that the magnitude of [Ca(2+)](i) oscillations is a direct result of extracellular calcium influx that is determined in part by action potential duration.

Development and characterization of an in vitro gingival epithelial model.

A 3-dimensional gingival epithelial model has been developed and characterized. Oral epithelial cells and connective tissue fibroblasts were isolated from human gingival tissue and used to create an in vitro oral mucosa co-culture model. Fibroblasts were seeded on a scaffold of nylon mesh, allowed to proliferate and secrete collagen and extracellular matrix proteins to form a stroma capable of supporting the growth of epithelial cells. Epithelial cells were seeded on top of a confluent stromal layer, proliferated and differentiated to form a stratified squamous epithelium. Resident epithelial cells were stimulated, by manipulation of growth medium and culture conditions, to form a multi-layered oral mucosa-like tissue. Histologic analyses revealed cellular architecture exhibiting stromal-epithelial interaction which supports the growth and differentiation of an epithelial layer. Immunohistochemical analyses confirmed production of types I and III collagen. Immunofluorescence of the stromal layer identified type IV collagen and fibronectin. Fibronectin was also detected on surface epithelium. Differentiation of basal, spinous and granular cells was observed, and the presence of differentiation markers, acidic (K10, 14-16, 19) and basic (K1-8) cytokeratins were confirmed using broad spectrum cytokeratin antibodies, AE1 and AE3. Development of a discontinuous basal lamina zone, with hemidesmosomes, was observed by electron microscopy. The co-culture was metabolically active, as measured by the thiazoyl blue (MTT) assay for mitchondrial function and [3H] thymidine incorporation into DNA. The human gingival epithelial co-culture model was viable up to 35 days post-epithelial seed. This model may offer opportunities for limited study of periodontal tissue responsiveness.

Beta IG-H3, a novel secretory protein inducible by transforming growth factor-beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro.

We have previously identified a gene, beta ig-h3, which is highly induced in A549 cells (human lung adenocarcinoma) after growth arrest by transforming growth factor-beta. The beta ig-h3 gene encodes a 683-amino-acid secretory protein termed beta IG-H3, and treatment of several cell lines with transforming growth factor-beta results in increased secretion of beta IG-H3 into cell culture supernatants. In this report, we further characterize beta IG-H3 with respect to its synthesis and function. Primary human foreskin fibroblasts grown in monolayer culture produced beta IG-H3 mRNA and secreted beta IG-H3 protein into the growth media. Treatment of these cells with transforming growth factor-beta led to an increase in beta IG-H3 mRNA and protein. Cells grown on three-dimensional scaffolds secreted beta IG-H3 into the extracellular matrix, as judged by immunostaining with anti-beta IG-H3 antibodies. beta IG-H3 was also detected in normal human skin, especially in the papillary dermis. Finally, we show that recombinant beta IG-H3 supported attachment and spreading of dermal fibroblasts, suggesting that beta IG-H3 may function as an extracellular attachment protein in skin.

Cartilage production by rabbit articular chondrocytes on polyglycolic acid scaffolds in a closed bioreactor system.

Rabbit articular chondrocytes were seeded onto three-dimensional polyglycolic acid (PGA) scaffolds and placed into a closed bioreactor system. After 4 weeks of growth, meshes were examined for cartilage formation. Gross examination revealed solid, glistening material that had the appearance of cartilaginous tissue. Histologic examination revealed cell growth and deposition of extracellular matrix throughout the mesh with a less dense central core. Alcian blue and Safranin 0 staining showed deposition of glycosaminoglycans (GAGs). Immunostaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen. Biochemical analysis showed collagen and GAG values to be 15% and 25% dry weight, respectively. Our results indicate that this type of system compares well with those previously described and should be useful for producing cartilage for evaluation in a clinical setting. (c) 1995 John Wiley & Sons, Inc.

Tgf-Beta promotes the growth of bovine chondrocytes in monolayer culture and the formation of cartilage tissue on three-dimensional scaffolds.

We have investigated the ability of transforming growth factor beta (TGF-beta) to promote the growth and differentiation of chondrocytes in monolayer and on three-dimensional scaffolds. Treatment of chondrocytes with TGF-beta and ascorbate individually stimulated the proliferation of bovine articular chondrocytes about 2-fold when cells were grown in monolayer culture: the combination of TGF-beta and ascorbate resulted in a 3-fold increase in cell number over a 72-h period. Peak stimulation with TGF-beta occurred at about 1.0 ng/ml: bFGF was slightly inhibitory in these assays. TGF-beta led to an increase in glycosaminoglycan synthesis as detected by Western blotting using anti-chondroitin sulfate antibodies. No significant change in collagen type II mRNA or protein was observed. When cells were grown on grown on three-dimensional scaffolds composed of polyglyocolic acid, TGF-beta treatment led to an increase in the size of the cartilage-like constructs produced. This was accompanied by increases in collagen and glycosaminoglycan deposition; immunohistochemical staining showed that the predominant collagen was type II. These results indicate that TGF-beta is capable of increasing the proliferation rate of chondrocytes in monolayer as well as increasing cartilage production on three-dimensional scaffolds and may find utility in the in vitro engineering of cartilage tissue.

Characterization, barrier function, and drug metabolism of an in vitro skin model.

We have characterized an in vitro skin model consisting of neonatal keratinocytes and fibroblasts grown on a nylon mesh. To produce a dermal model, fibroblasts were seeded onto nylon mesh and grown for 4 weeks until a physiologic dermal-like matrix was formed. This matrix was found to consist of collagens I and III, fibronectin, and glycosaminoglycans. Keratinocytes were then seeded onto the dermal model and the co-culture was grown at the air/liquid interface. A differentiated epidermis with distinct basal, spinous, granular, and stratum corneum layers was formed. When incubated in the presence of keratinocytes, fibronectin immunofluorescence increased throughout the dermis compared to cultures incubated similarly in the absence of keratinocytes. A basement membrane zone rich in laminin, collagen IV, and heparan sulfate proteoglycan was detected. The epidermis, isolated from the co-culture by thermolysin digestion, was analyzed for differentiation markers. K1 keratin (67-kDa) and involucrin were detected by immunologic techniques. Ceramide lipids (types III and IV), thought to be important in barrier function, were detected by thin-layer chromatography. The permeability of the co-culture to a panel of compounds, including [3H]-water, was determined using Franz and side-by-side diffusion cells. The permeability coefficient for water was of the same order of magnitude as that determined for neonatal foreskin. The co-culture also showed selective permeability to a panel of compounds of differing lipid solubility. This co-culture metabolized [3H]-testosterone to a profile of metabolites similar to that of neonatal foreskin. We believe that this in vitro skin model will be useful for the study of drug permeability and metabolism.