High Hydrostatic Pressure Therapy Annihilates Squamous Cell Carcinoma in a Murine Model

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers. In the treatment of cSCC, it is necessary to remove it completely, and reconstructive surgery, such as a skin graft or a local or free flap, will be required, depending on the size, with donor-site morbidity posing a burden to the patient. The high hydrostatic pressure (HHP) technique has been developed as a physical method of decellularizing various tissues. We previously reported that HHP at 200 MPa for 10 min could inactivate all cells in the giant congenital melanocytic nevus, and we have already started a clinical trial using this technique. In the present study, we explored the critical pressurization condition for annihilating cSCC cells in vitro and confirmed that this condition could also annihilate cSCC in vivo. We prepared 5 pressurization conditions in this study (150, 160, 170, 180, and 190 MPa for 10 min) and confirmed that cSCC cells were killed by pressurization at ≥160 MPa for 10 min. In the in vivo study, the cSCC cells inactivated by HHP at 200 MPa for 10 min were unable to proliferate after injection into the intradermal space of mice, and transplanted cSCC tissues that had been inactivated by HHP showed a decreased weight at 5 weeks after implantation. These results suggested that HHP at 200 MPa for 10 min was able to annihilate SCC, so HHP technology may be a novel treatment of skin cancer

Exploration of the Pressurization Condition for Killing Human Skin Cells and Skin Tumor Cells by High Hydrostatic Pressure

High hydrostatic pressure (HHP) is a physical method for inactivating cells or tissues without using chemicals such as detergents. We previously reported that HHP at 200 MPa for 10 min was able to inactivate all cells in skin and giant congenital melanocytic nevus (GCMN) without damaging the extracellular matrix. We also reported that HHP at 150 MPa for 10 min was not sufficient to inactivate them completely, while HHP at 200 MPa for 10 min was able to inactivate them completely. We intend to apply HHP to treat malignant skin tumor as the next step; however, the conditions necessary to kill each kind of cell have not been explored. In this work, we have performed a detailed experimental study on the critical pressure and pressurization time using five kinds of human skin cells and skin tumor cells, including keratinocytes (HEKas), dermal fibroblasts (HDFas), adipose tissue-derived stem cells (ASCs), epidermal melanocytes (HEMa-LPs), and malignant melanoma cells (MMs), using pressures between 150 and 200 MPa. We pressurized cells at 150, 160, 170, 180, or 190 MPa for 1 s, 2 min, and 10 min and evaluated the cellular activity using live/dead staining and proliferation assays. The proliferation assay revealed that HEKas were inactivated at a pressure higher than 150 MPa and a time period longer than 2 min, HDFas and MMs were inactivated at a pressure higher than 160 MPa and for 10 min, and ASCs and HEMa-LPs were inactivated at a pressure higher than 150 MPa and for 10 min. However, some HEMa-LPs were observed alive after HHP at 170 MPa for 10 min, so we concluded that HHP at a pressure higher than 180 MPa for 10 min was able to inactivate five kinds of cells completely

Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples

Collagen/Gelatin Sponges (CGSs) Provide Both Protection and Release of bFGF: An In Vitro Study

It has been reported that collagen/gelatin sponges (CGSs) are able to sustain the release of basic fibroblast growth factor (bFGF) for approximately 10 days via the formation of ion complexes between bFGF and gelatin. CGSs impregnated with bFGF have been proven to promote dermis-like tissue formation in various in vivo studies and clinical trials. However, the bioactivities of bFGF released from CGSs have not been explored in vitro. In this study, we explored the ability of CGS impregnated with bFGF, stored at 37°C for up to 14 days, to promote fibroblast proliferation and the sustained release of bFGF. We analyzed the cellular viability and proliferation in 2D and in 3D cell cultures, by a CCK-8 assay. Furthermore, in order to characterize the morphological alteration of fibroblasts, we studied 3D cultures by microscopy with a scanning electron microscope (SEM) and a confocal microscope. Our analyses revealed that the fibroblasts were elongated and flanked each other. They infiltrated and migrated inside the CGSs and were oriented along the CGS structure. Thus, these data prove that CGSs protect and sustain the efficient release of growth factor for more than 7 days

Evaluation of adipogenesis over time using a novel bioabsorbable implant without the addition of exogenous cells or growth factors

Background: Breast reconstruction is crucial for patients who have undergone mastectomy for breast cancer. Our bioabsorbable implants comprising an outer poly-l-lactic acid mesh and an inner component filled with collagen sponge promote and retain adipogenesis in vivo without the addition of exogenous cells or growth factors. In this study, we evaluated adipogenesis over time histologically and at the gene expression level using this implant in a rodent model. Methods: The implants were inserted in the inguinal and dorsal regions of the animals. At 1, 3, 6, and 12 months post-operation, the weight, volume, and histological assessment of all newly formed tissue were performed. We analyzed the formation of new adipose tissue using multiphoton microscopy and RNA sequencing. Results: Both in the inguinal and dorsal regions, adipose tissue began to form 1 month post-operation in the peripheral area. Angiogenesis into implants was observed until 3 months. At 6 months, microvessels matured and the amount of newly generated adipose tissue peaked and was uniformly distributed inside implants. The amount of newly generated adipose tissue decreased from 6 to 12 months but at 12 months, adipose tissue was equivalent to the native tissue histologically and in terms of gene expression. Conclusions: Our bioabsorbable implants could induce normal adipogenesis into the implants after subcutaneous implantation. Our implants can serve as a novel and safe material for breast reconstruction without requiring exogenous cells or growth factors

Hydrostatic pressure can induce apoptosis of the skin

Abstract We previously showed that high hydrostatic pressure (HHP) treatment at 200 MPa for 10 min induced complete cell death in skin and skin tumors via necrosis. We used this technique to treat a giant congenital melanocytic nevus and reused the inactivated nevus tissue as a dermis autograft. However, skin inactivated by HHP promoted inflammation in a preclinical study using a porcine model. Therefore, in the present study, we explored the pressurization conditions that induce apoptosis of the skin, as apoptotic cells are not believed to promote inflammation, so the engraftment of inactivated skin should be improved. Using a human dermal fibroblast cell line in suspension culture, we found that HHP at 50 MPa for ≥ 36 h completely induced fibroblast cell death via apoptosis based on the morphological changes in transmission electron microscopy, reactive oxygen species elevation, caspase activation and phosphatidylserine membrane translocation. Furthermore, immunohistochemistry with terminal deoxynucleotidyl transferase dUTP nick-end labeling and cleaved caspase-3 showed most cells in the skin inactivated by pressurization to be apoptotic. Consequently, in vivo grafting of apoptosis-induced inactivated skin resulted in successful engraftment and greater dermal cellular density and macrophage infiltration than our existing method. Our finding supports an alternative approach to hydrostatic pressure application