Caroline Weinstein-Oppenheimer

The effect of an autologous cellular gel-matrix integrated implant system on wound healing

BackgroundThis manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions.MethodsThe best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion.ResultsThe preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate (p = 0,06).ConclusionIIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties.

The effects of beta-estradiol on Raf activity, cell cycle progression and growth factor synthesis in the MCF-7 breast cancer cell line.

The aim of this study was to test the hypothesis that some of the proliferative effects of steroid hormones on cancer cells are mediated by the Raf proto-oncogenes. The human breast cancer cell line MCF-7 is estrogen-receptor (ER) positive (+). NCI/ADR-RES is a human cell line lacking the estrogen receptor (ER-) that was initially named MCF-ADR. Raf-1, A-Raf and B-Raf kinase activities were examined in cell lines treated with b-estradiol for 24 hours. Increases in Raf-1 and A-Raf activities were observed after treatment with b-estradiol in the ER (+) MCF-7 cells but not in the ER (-) NCI/ADR-RES cells. In contrast, no significant changes in B-Raf activity were observed. Thus b-estradiol can induce Raf-1 and A-Raf activities in ER (+) cells. In addition, b-estradiol caused cell cycle progression in MCF-7 cells and an increased proliferative response to b-estradiol was observed in MCF-7, which overexpressed constitutively-active Raf-1 (MCF/DRaf-1). Increased mRNA levels of the ligand for the c-erb-B2 receptor, amphiregulin (ARG) were observed after b-estradiol treatment of MCF-7 cells whereas constitutively higher levels of ARG and its receptor, c-erb-B2 mRNAs were detected in MCF/DRaf-1 cells. These findings suggest that targeting Raf may prove efficacious in breast cancer therapies.

Designing a gelatin/chitosan/hyaluronic acid biopolymer using a thermophysical approach for use in tissue engineering.

Cell culture on biopolymeric scaffolds has provided treatments for tissue engineering. Biopolymeric mixtures based on gelatin (Ge), chitosan (Ch) and hyaluronic acid (Ha) have been used to make scaffolds for wound healing. Thermal and physical properties of scaffolds prepared with Ge, Ch and Ha were characterized. Thermal characterization was made by using differential scanning calorimetry (DSC), and physical characterization by gas pycnometry and scanning electron microscopy. The effects of Ge content and cross-linking on thermophysical properties were evaluated by means of a factorial experiment design (central composite face centered). Gelatin content was the main factor that affects the thermophysical properties (microstructure and thermal transitions) of the scaffold. The effect of Ge content of the scaffolds for tissue engineering was studied by seeding skin cells on the biopolymers. The cell attachment was not significantly modified at different Ge contents; however, the cell growth rate increased linearly with the decrease of the Ge content. This relationship together with the thermophysical characterization may be used to design scaffolds for tissue engineering.

Growth factor production from fibrin-encapsulated human keratinocytes.

Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-β1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-β1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-β1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-β1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.

Effect of the standardized Cimicifuga foetida extract on Hsp 27 expression in the MCF-7 cell line

Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.

Design of a hybrid biomaterial for tissue engineering: Biopolymer-scaffold integrated with an autologous hydrogel carrying mesenchymal stem-cells

Biologically active biomaterials as biopolymers and hydrogels have been used in medical applications providing favorable results in tissue engineering. In this research, a wound dressing device was designed by integration of an autologous clot hydrogel carrying mesenchymal stem-cells onto a biopolymeric scaffold. This hybrid biomaterial was tested in-vitro and in-vivo, and used in a human clinical case. The biopolymeric scaffold was made with gelatin, chitosan and hyaluronic acid, using a freeze-drying method. The scaffold was a porous material which was designed evaluating both physical properties (glass transition, melting temperature and pore size) and biological properties (cell viability and fibronectin expression). Two types of chitosan (120 and 300kDa) were used to manufacture the scaffold, being the high molecular weight the most biologically active and stable after sterilization with gamma irradiation (25kGy). A clot hydrogel was formulated with autologous plasma and calcium chloride, using an approach based on design of experiments. The optimum hydrogel was used to incorporate cells onto the porous scaffold, forming a wound dressing biomaterial. The wound dressing device was firstly tested in-vitro using human cells, and then, its biosecurity was evaluated in-vivo using a rabbit model. The in-vitro results showed high cell viability after one week (99.5%), high mitotic index (19.8%) and high fibronectin expression. The in-vivo application to rabbits showed adequate biodegradability capacity (between 1 and 2weeks), and the histological evaluation confirmed absence of rejection signs and reepithelization on the wound zone. Finally, the wound dressing biomaterial was used in a single human case to implant autologous cells on a skin surgery. The medical examination indicated high biocompatibility, partial biodegradation at one week, early regeneration capacity at 4weeks and absence of rejection signs.

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright

Edible scaffolds based on non-mammalian biopolymers for myoblast growth

In vitro meat has recently emerged as a new concept in food biotechnology. Methods to produce in vitro meat generally involve the growth of muscle cells that are cultured on scaffolds using bioreactors. Suitable scaffold design and manufacture are critical to downstream culture and meat production. Most current scaffolds are based on mammalian-derived biomaterials, the use of which is counter to the desire to obviate mammal slaughter in artificial meat production. Consequently, most of the knowledge is related to the design and control of scaffold properties based on these mammalian-sourced materials. To address this, four different scaffold materials were formulated using non-mammalian sources, namely, salmon gelatin, alginate, and additives including gelling agents and plasticizers. The scaffolds were produced using a freeze-drying process, and the physical, mechanical, and biological properties of the scaffolds were evaluated. The most promising scaffolds were produced from salmon gelatin, alginate, agarose, and glycerol, which exhibited relatively large pore sizes (~200 μm diameter) and biocompatibility, permitting myoblast cell adhesion (~40%) and growth (~24 h duplication time). The biodegradation profiles of the scaffolds were followed, and were observed to be less than 25% after 4 weeks. The scaffolds enabled suitable myogenic response, with high cell proliferation, viability, and adequate cell distribution throughout. This system composed of non-mammalian edible scaffold material and muscle-cells is promising for the production of in vitro meat.

Synthesis of chalcones with antiproliferative activity on the SH-SY5Y neuroblastoma cell line: Quantitative Structure–Activity Relationship Models

Chalcones are a group of molecules with a broad spectrum of biological activities, being especially appealing for their antiproliferative effects on several cancer cell lines. For this reason, we synthesized 23 chalcones with good to excellent yields and assessed their effect on the viability of the SH-SY5Y neuroblastoma cell line and on primary human fibroblasts. The results indicated that 18 of these compounds were more active than 5-fluorouracil in the cancer cell line and one of them was more selective than this reference drug. To identify structural features related to the antiproliferative activity of these compounds, as well as, the selectivity on the cancer cell line, a 2D-QSAR analysis was performed. The QSAR model (q2 = 0.803; r2 = 0.836) showed that lipophilicity (CLogP) is the most important factor to increase their cytotoxicity on the cancer cell line. On the other hand, the selectivity QSAR model (q2 = 0.917; r2 = 0.916) showed that changes in the Mulliken’s charge of the carbonyl group and at the C4’ position in the chalcone core can increase the selectivity for SH-SY5Y cell line compared to normal fibroblasts.