Subcutis Of Nude Mice Research Articles

Inactivated Nevus Tissue with High Hydrostatic Pressure Treatment Used as a Dermal Substitute after a 28-Day Cryopreservation Period.

Background Giant congenital melanocytic nevi (GCMN) treatment remains controversial. While surgical resection is the best option for complete removal, skin shortage to reconstruct the skin defect remains an issue. We report a novel treatment using a high hydrostatic pressurization (HHP) technique and a cryopreservation procedure. However, cryopreservation may inhibit revascularization of implanted nevus tissue and cultured epidermal autograft (CEA) take. We aimed to investigate the influence of the cryopreservation procedure on the HHP-treated dermis specimen and CEA take on cryopreserved tissue. Methods Nevus tissue harvested from a patient with GCMN was inactivated with HHP of 200 MPa and then cryopreserved at -30°C for 28 days. The cryopreserved specimen was compared with fresh (HHP-treated without cryopreservation) tissue and with untreated (without HHP treatment) tissue to evaluate the extracellular matrix, basal membranes, and capillaries. Cultured epidermis (CE) take on the cryopreserved tissue was evaluated following implantation of the cryopreserved nevus tissue with CE into the subcutis of nude mice. Results No difference was observed between cryopreserved and fresh tissue in terms of collagen or elastic fibers, dermal capillaries, or basement membranes at the epidermal-dermal junction. In 4 of 6 samples (67%), applied CE took on the nevus tissues and regenerated the epidermis in the cryopreserved group compared with 5 of 6 samples (83%) in the fresh group. Conclusion Cryopreservation at -30°C for 28 days did not result in significant damage to inactivated nevus tissue, and applied CE on the cryopreserved nevus tissues took and regenerated the epidermis. Inactivated nevus tissue with HHP can be used as a dermal substitute after 28-day cryopreservation.

Silanization of Chitosan and Hydrogel Preparation for Skeletal Tissue Engineering.

Tissue engineering is a multidisciplinary field that relies on the development of customized biomaterial to support cell growth, differentiation and matrix production. Toward that goal, we designed the grafting of silane groups onto the chitosan backbone (Si-chito) for the preparation of in situ setting hydrogels in association with silanized hydroxypropyl methylcellulose (Si-HPMC). Once functionalized, the chitosan was characterized, and the presence of silane groups and its ability to gel were demonstrated by rheology that strongly suggests the presence of silane groups. Throughout physicochemical investigations, the Si-HPMC hydrogels containing Si-chito were found to be stiffer with an injection force unmodified. The presence of chitosan within the hydrogel has demonstrated a higher adhesion of the hydrogel onto the surface of tissues. The results of cell viability assays indicated that there was no cytotoxicity of Si-chito hydrogels in 2D and 3D culture of human SW1353 cells and human adipose stromal cells, respectively. Moreover, Si-chito allows the transplantation of human nasal chondrocytes in the subcutis of nude mice while maintaining their viability and extracellular matrix secretory activity. To conclude, Si-chito mixed with Si-HPMC is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC.

A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs.

Articular cartilage (AC) may be affected by many injuries including traumatic lesions that predispose to osteoarthritis. Currently there is no efficient cure for cartilage lesions. In that respect, new strategies for regenerating AC are contemplated with interest. In this context, we aim to develop and characterize an injectable, self-hardening, mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropymethyl cellulose (Si-HPMC) mixed with silanised chitosan. The in vitro cytocompatibility of Si-HPCH was tested using human adipose stromal cells (hASC). In vivo, we first mixed Si-HPCH with hASC to observe cell viability after implantation in nude mice subcutis. Si-HPCH associated or not with canine ASC (cASC), was then tested for the repair of osteochondral defects in canine femoral condyles. Our data demonstrated that Si-HPCH supports hASC viability in culture. Moreover, Si-HPCH allows the transplantation of hASC in the subcutis of nude mice while maintaining their viability and secretory activity. In the canine osteochondral defect model, while the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without cASC, revealed a significant osteochondral regeneration. To conclude, Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC as well as the regeneration of osteochondral defects in dogs when implanted alone or with ASC.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Antitumor effect of palmitic acid-conjugated DsiRNA for colon cancer in a mouse subcutaneous tumor model.

In this study, we synthesized Dicer-substrate siRNA conjugated with palmitic acid at the 5'-end of the sense strand (C16-DsiRNA), and examined its RNAi effect on β-catenin as a target gene in a colon cancer cell line, HT29Luc, both in vitro and in vivo. We examined the in vitro RNAi effect in HT29Luc cells and found that C16-DsiRNA strongly inhibited expression of the β-catenin gene in comparison with non-modified DsiRNA. Also, high membrane permeability of C16-DsiRNA was exhibited, and it was confirmed that most of the C16-DsiRNA was localized in cytoplasm of HT29Luc cells. In regard to the in vivo RNAi effect, C16-DsiRNA complexed with Invivofectamine targeting the β-catenin gene was locally administered to a subcutaneous tumor formed by implantation of HT29Luc cells into the subcutis of nude mice; we evaluated the effect by measuring the bioluminescence increase, which reflects tumor growth, using an in vivo imaging system. As a result, C16-DsiRNA strongly inhibited the growth of tumors formed in subcutis of nude mice compared with non-modified DsiRNA, and this in vivo RNAi effect lasted up to 15days. Our results suggest that C16-DsiRNA should be vigorously pursued as a novel nucleic acid medicine for clinical treatment of cancer.

Overexpression of secretory phospholipase A2-IIa supports cancer stem cell phenotype via HER/ERBB-elicited signaling in lung and prostate cancer cells.

Resistance to conventional chemotherapies remains a significant clinical challenge in treatment of cancer. The cancer stem cells (CSCs) have properties necessary for tumor initiation, resistance to therapy, and progression. HER/ERBB‑elicited signaling supports CSC properties. Our previous studies revealed that secretory phospholipaseA2 groupIIa (sPLA2‑IIa) is overexpressed in both prostate and lung cancer cells, leading to an aberrant high level in the interstitial fluid, i.e.,tumor microenvironment and blood. HER/ERBB-PI3K-Akt-NF-κB signaling stimulates sPLA2‑IIa overexpression, and in turn, sPLA2‑IIa activates EGFR family receptors and HER/ERBB-elicited signaling and stimulates sPLA2‑IIa overexpression in a positive feedback manner. The present study determined the molecular mechanisms of sPLA2‑IIa in stimulating HER/ERBB-elicited signaling and supporting CSC properties. We found that sPLA2‑IIa binds both EGFR and HER3 demonstrated by co-immunoprecipitation experiments and also indirectly interacts with HER2, suggesting that sPLA2‑IIa functions as a ligand for both EGFR and HER3. Furthermore, both side population CSCs from non-small cell lung cancer (NSCLC) A549 and H1975 cells and ALDH1‑high CSCs from castration-resistant prostate cancer (CRPC) 22Rv1 cells overexpress sPLA2‑IIa and produce tumors when inoculated into subcutis of nude mice. Given an aberrant high level of sPLA2‑IIa in the tumor microenvironment that should be much higher than that in the blood, our findings support the notion that sPLA2‑IIa functions as a ligand for EGFR family receptors and supports CSC properties via HER/ERBB-elicited signaling, which may contribute to resistance to therapy and cancer progression.

Bevacizumab-Induced Inhibition of Angiogenesis Promotes a More Homogeneous Intratumoral Distribution of Paclitaxel, Improving the Antitumor Response

The antitumor activity of angiogenesis inhibitors is reinforced in combination with chemotherapy. It is debated whether this potentiation is related to a better drug delivery to the tumor due to the antiangiogenic effects on tumor vessel phenotype and functionality. We addressed this question by combining bevacizumab with paclitaxel on A2780-1A9 ovarian carcinoma and HT-29 colon carcinoma transplanted ectopically in the subcutis of nude mice and on A2780-1A9 and IGROV1 ovarian carcinoma transplanted orthotopically in the bursa of the mouse ovary. Paclitaxel concentrations together with its distribution by MALDI mass spectrometry imaging (MALDI MSI) were measured to determine the drug in different areas of the tumor, which was immunostained to depict vessel morphology and tumor proliferation. Bevacizumab modified the vessel bed, assessed by CD31 staining and dynamic contrast enhanced MRI (DCE-MRI), and potentiated the antitumor activity of paclitaxel in all the models. Although tumor paclitaxel concentrations were lower after bevacizumab, the drug distributed more homogeneously, particularly in vascularized, non-necrotic areas, and was cleared more slowly than controls. This happened specifically in tumor tissue, as there was no change in paclitaxel pharmacokinetics or drug distribution in normal tissues. In addition, the drug concentration and distribution were not influenced by the site of tumor growth, as A2780-1A9 and IGROV1 growing in the ovary gave results similar to the tumor growing subcutaneously. We suggest that the changes in the tumor microenvironment architecture induced by bevacizumab, together with the better distribution of paclitaxel, may explain the significant antitumor potentiation by the combination.

The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization.

We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP) higher than 200 MPa and that human cultured epidermis (hCE) engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa). Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM). hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting.

Inactivation of Human Nevus Tissue Using High Hydrostatic Pressure for Autologous Skin Reconstruction: A Novel Treatment for Giant Congenital Melanocytic Nevi

Giant congenital melanocytic nevi are intractable lesions associated with a risk of melanoma. High hydrostatic pressure (HHP) technology is a safe physical method for producing decellularized tissues without chemicals. We have reported that HHP can inactivate cells present in various tissues without damaging the native extracellular matrix (ECM). The objectives of this study were to inactivate human nevus tissue using HHP and to explore the possibility of reconstructing skin using inactivated nevus in combination with cultured epidermis (CE). Human nevus specimens 8 mm in diameter were pressurized by HHP at 100, 200, 500, and 1000 MPa for 10 min. The viability of specimens just after HHP, outgrowth of cells, and viability after cultivation were evaluated to confirm the inactivation by HHP. Histological evaluation using hematoxylin-eosin staining and immunohistochemical staining for type IV collagen was performed to detect damage to the ECM of the nevus. The pressurized nevus was implanted into the subcutis of nude mice for 6 months to evaluate the retention of human cells. Then, human CE was applied on the pressurized nevus and implanted into the subcutis of nude mice. The viability of pressurized nevus was not detected just after HHP and after cultivation, and outgrowth of fibroblasts was not observed in the 200, 500, and 1000 MPa groups. Human cells were not observed after 6 months of implantation in these groups. No apparent damage to the ECM was detected in all groups; however, CE took on nevus in the 200 and 500 MPa groups, but not in the 1000 MPa group. These results indicate that human nevus tissue was inactivated by HHP at more than 200 MPa; however, HHP at 1000 MPa might cause damage that prevents the take of CE. In conclusion, all cells in nevus specimens were inactivated after HHP at more than 200 MPa and this inactivated nevus could be used as autologous dermis for covering full-thickness skin defects after nevus removal. HHP between 200 and 500 MPa will be optimal to reconstruct skin in combination with cultured epidermal autograft without damage to the ECM.

Preparation of Inactivated Human Skin Using High Hydrostatic Pressurization for Full-Thickness Skin Reconstruction.

We have reported that high-hydrostatic-pressure (HHP) technology is safe and useful for producing various kinds of decellularized tissue. However, the preparation of decellularized or inactivated skin using HHP has not been reported. The objective of this study was thus to prepare inactivated skin from human skin using HHP, and to explore the appropriate conditions of pressurization to inactivate skin that can be used for skin reconstruction. Human skin samples of 8 mm in diameter were packed in bags filled with normal saline solution (NSS) or distilled water (DW), and then pressurized at 0, 100, 150, 200 and 1000 MPa for 10 minutes. The viability of skin after HHP was evaluated using WST-8 assay. Outgrowth cells from pressurized skin and the viability of pressurized skin after cultivation for 14 days were also evaluated. The pressurized skin was subjected to histological evaluation using hematoxylin and eosin staining, scanning electron microscopy (SEM), immunohistochemical staining of type IV collagen for the basement membrane of epidermis and capillaries, and immunohistochemical staining of von Willebrand factor (vWF) for capillaries. Then, human cultured epidermis (CE) was applied on the pressurized skin and implanted into the subcutis of nude mice; specimens were subsequently obtained 14 days after implantation. Skin samples pressurized at more than 200 MPa were inactivated in both NSS and DW. The basement membrane and capillaries remained intact in all groups according to histological and immunohistological evaluations, and collagen fibers showed no apparent damage by SEM. CE took on skin pressurized at 150 and 200 MPa after implantation, whereas it did not take on skin pressurized at 1000 MPa. These results indicate that human skin could be inactivated after pressurization at more than 200 MPa, but skin pressurized at 1000 MPa had some damage to the dermis that prevented the taking of CE. Therefore, pressurization at 200 MPa is optimal for preparing inactivated skin that can be used for skin reconstruction.

Enriching a cellulose hydrogel with a biologically active marine exopolysaccharide for cell-based cartilage engineering.

The development of biologically and mechanically competent hydrogels is a prerequisite in cartilage engineering. We recently demonstrated that a marine exopolysaccharide, GY785, stimulates the in vitro chondrogenesis of adipose stromal cells. In the present study, we thus hypothesized that enriching our silated hydroxypropyl methylcellulose hydrogel (Si-HPMC) with GY785 might offer new prospects in the development of scaffolds for cartilage regeneration. The interaction properties of GY785 with growth factors was tested by surface plasmon resonance (SPR). The biocompatibility of Si-HPMC/GY785 towards rabbit articular chondrocytes (RACs) and its ability to maintain and recover a chondrocytic phenotype were then evaluated in vitro by MTS assay, cell counting and qRT-PCR. Finally, we evaluated the potential of Si-HPMC/GY785 associated with RACs to form cartilaginous tissue in vivo by transplantation into the subcutis of nude mice for 3 weeks. Our SPR data indicated that GY785 was able to physically interact with BMP-2 and TGFβ. Our analyses also showed that three-dimensionally (3D)-cultured RACs into Si-HPMC/GY785 strongly expressed type II collagen (COL2) and aggrecan transcripts when compared to Si-HPMC alone. In addition, RACs also produced large amounts of extracellular matrix (ECM) containing glycosaminoglycans (GAG) and COL2. When dedifferentiated RACs were replaced in 3D in Si-HPMC/GY785, the expressions of COL2 and aggrecan transcripts were recovered and that of type I collagen decreased. Immunohistological analyses of Si-HPMC/GY785 constructs transplanted into nude mice revealed the production of a cartilage-like extracellular matrix (ECM) containing high amounts of GAG and COL2. These results indicate that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Establishment of a novel Chinese metastatic melanoma cell line showing the new cytogenetic and biological properties.

Malignant melanoma remains the most life-threatening skin cancer to date. What makes it worse is the incidence keeps increasing worldwide, including in China. Notably, clinical studies revealed the distinct features in the Chinese population differing from those in Caucasians, which give hints to variant mechanisms underlying. Therefore, it is of great importance to generate a cell line with similar background for melanoma research in Chinese even Asian patients. However, most melanoma cell lines in use are derived from Caucasians, thus, we established one novel metastatic melanoma cell line, FLFMM-34, derived from a Han Chinese woman. The cell line showed positive for S100, HMB45, vimentin and melan-A. Chromosome analysis revealed multiple structural aberrations. Gene-mutation analysis identified that FLFMM-34 cells had BRAF(V600E) mutation and deletions of exon 2 and 3 in p16/CDKN2A. Importantly, two novel mutations including TP53(P33R) and TP53(R142H) have been detected. RT-PCR results showed that FLFMM-34 cells expressed a higher mRNA level of cyclinD1 than three other melanoma cell lines, WM793B, 1205Lu and A2058. In addition, in vivo mice model demonstrated that the cells could be transplanted into the subcutis of nude mice and produced tumors associated with lymphoid node metastases. In conclusion, these data indicate that FLFMM-34 cell line can be employed as a suitable model for melanoma research in Chinese Han population.

Critical role of histone demethylase RBP2 in human gastric cancer angiogenesis

BackgroundThe molecular mechanisms responsible for angiogenesis and abnormal expression of angiogenic factors in gastric cancer, including vascular endothelial growth factor (VEGF), remain unclear. The histone demethylase retinoblastoma binding protein 2 (RBP2) is involved in gastric tumorgenesis by inhibiting the expression of cyclin-dependent kinase inhibitors (CDKIs).MethodsThe expression of RBP2, VEGF, CD31, CD34 and Ki67 was assessed in 30 human gastric cancer samples and normal control samples. We used quantitative RT-PCR, western blot analysis, ELISA, tube-formation assay and colony-formation assay to characterize the change in VEGF expression and associated biological activities induced by RBP2 silencing or overexpression. Luciferase assay and ChIP were used to explore the direct regulation of RBP2 on the promoter activity of VEGF. Nude mice and RBP2-targeted mutant mice were used to detect the role of RBP2 in VEGF expression and angiogenesis in vivo.ResultsRBP2 and VEGF were both overexpressed in human gastric cancer tissue, with greater microvessel density (MVD) and cell proliferation as compared with normal tissue. In gastric epithelial cell lines, RBP2 overexpression significantly promoted the expression of VEGF and the growth and angiogenesis of the cells, while RBP2 knockdown had the reverse effect. RBP2 directly bound to the promoter of VEGF to regulate its expression by histone H3K4 demethylation. The subcutis of nude mice transfected with BGC-823 cells with RBP2 knockdown showed reduced VEGF expression and MVD, with reduced carcinogenesis and cell proliferation. In addition, the gastric epithelia of RBP2 mutant mice with increased H3K4 trimethylation showed reduced VEGF expression and MVD.ConclusionsThe promotion of gastric tumorigenesis by RBP2 was significantly associated with transactivation of VEGF expression and elevated angiogenesis. Overexpression of RBP2 and activation of VEGF might play important roles in human gastric cancer development and progression.

Establishment and characterization of a cell line (NOMH-1) originating from a human endometrioid adenocarcinoma of the ovary

BackgroundCell lines are very useful for clinical and basic research. Thus far, only 11 reports have documented the characteristics of ovarian endometrioid adenocarcinoma cell lines in the literature. Due to the scarcity of information, the establishment of an ovarian malignant tumor cell line with distinctive characteristics is particularly important to study this disease. Thus, this study was undertaken to establish and characterize a new human endometrioid adenocarcinoma cell line of the ovary.MethodsThe cell line NOMH-1 was established from an ovarian tumor of a 44-year-old woman. Features of the cell line studied included morphology, chromosome analysis, heterotransplantation, tumor markers, and chemosensitivity.ResultsThis cell line has been growing well for 232 months and subcultured more than 50 times. Monolayer cultured cells were polygonal in shape, showing a pavement-like arrangement and a tendency to stack without contact inhibition. They exhibited a human karyotype with a modal chromosomal number in the hypertriploid range. The cells could be transplanted into the subcutis of nude mice and produced tumors resembling the original tumor. NOMH-1 cells expressed both CEA and CA19-9 which were identified immunohistochemically in the original tumor and the heterotransplanted tumor. The cells were sensitive to paclitaxel, an agent commonly used in the treatment of gynecological cancers.ConclusionsNOMH-1 is the first ovarian endometrioid adenocarcinoma cell line in which CEA and CA19-9 expression have been defined. This newly established cell line should be useful for investigating the characteristics of ovarian endometrioid adenocarcinoma.

Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor

We have developed a collagen/gelatin sponge (CGS) that can provide a sustained release of basic fibroblast growth factor (bFGF). In our previous study, it was shown that CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation two or three times earlier than an existing collagen sponge. In this study, adipogenesis was evaluated using CGSs disseminated with adipose tissue-derived stem cells (ASCs). Human ASCs were primarily isolated from human adipose tissue that was obtained during breast cancer surgery with informed consent at Kyoto University Hospital. ASCs were isolated from collagenase digests of adipose tissue. ASCs were labelled with PKH26. CGSs (8 mm diameter × 3 mm thickness) were impregnated with bFGF (0.1, 1, 7, 14 µg/cm(2) ) or normal saline solution. Then the labelled cells were disseminated (passage 3) on CGSs at a seeding density of 1 × 10(5) cells/cm(2) and implanted into the back subcutis of nude mice. Six weeks after implantation, adipogenesis at the administered site was evaluated. Immunohistological staining with von Willebrand factor (vWf) was performed to evaluate newly formed capillaries. Newly formed adipose tissue was observed macroscopically and histologically in all groups. The weight and area of regenerated adipose tissue were largest in the 1 µg/cm(2) bFGF group. Under a fluorescent microscope, newly formed adipose tissue in the bFGF-administered group was PKH-positive. These findings show that ASCs differentiated and formed adipose tissue. In this study, we showed that our CGSs impregnated with bFGF could be used as scaffolds with ASCs for adipogenesis.

Osteoblastic differentiation and potent osteogenicity of three-dimensional hBMSC-BCP particle constructs

Bone tissue engineering usually consists of associating osteoprogenitor cells and macroporous scaffolds. This study investigated the in vitro osteoblastic differentiation and resulting in vivo bone formation induced by a different approach that uses particles as substrate for human bone marrow stromal cells (hBMSCs), in order to provide cells with a higher degree of freedom and allow them to synthesize a three-dimensional (3D) environment. Biphasic calcium phosphate (BCP) particles (35 mg, ~175 µm in diameter) were therefore associated with 4 × 10(5) hBMSCs. To discriminate the roles of BCP properties and cell-synthesized 3D environments, inert glass beads (GBs) of similar size were used under the same conditions. In both cases, high cell proliferation and extensive extracellular matrix (ECM) production resulted in the rapid formation of thick cell-synthesized 3D constructs. In vitro, spontaneous osteoblastic differentiation was observed in the 3D constructs at the mRNA and protein levels by monitoring the expression of Runx2, BMP2, ColI, BSP and OCN. The hBMSC-BCP particle constructs implanted in the subcutis of nude mice induced abundant ectopic bone formation after 8 weeks (~35%, n = 5/5). In comparison, only fibrous tissue without bone was observed in the implanted hBMSC-GB constructs (n = 0/5). Furthermore, little bone formation (~3%, n = 5/5) was found in hBMSC-macroporous BCP discs (diameter 8 × 3 mm). This study underlines the lack of correspondence between bone formation and in vitro differentiation assays. Furthermore, these results highlight the importance of using BCP as well as a 3D environment for achieving high bone yield of interest for bone engineering.

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice

Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

Development of a brain metastatic canine prostate cancer cell line

Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer. A new cell line (Leo) was derived from a dog with spontaneous prostate cancer. Immunohistochemistry and PCR were used to characterize the primary prostate cancer and xenografts in nude mice. Subcutaneous tumor growth and metastases in nude mice were evaluated by bioluminescent imaging, radiography and histopathology. In vitro chemosensitivity of Leo cells to therapeutic agents was measured. Leo cells expressed the secretory epithelial cytokeratins (CK)8, 18, and ductal cell marker, CK7. The cell line grew in vitro (over 75 passages) and was tumorigenic in the subcutis of nude mice. Following intracardiac injection, Leo cells metastasized to the brain, spinal cord, bone, and adrenal gland. The incidence of metastases was greatest to the central nervous system (80%) with a lower incidence to bone (20%) and the adrenal glands (16%). In vitro chemosensitivity assays demonstrated that Leo cells were sensitive to Velcade and an HDAC-42 inhibitor with IC(50) concentrations of 1.9 nm and 0.95 µm, respectively. The new prostate cancer cell line (Leo) will be a valuable model to investigate the mechanisms of the brain and bone metastases.

Consistent Osteoblastic Differentiation of Human Mesenchymal Stem Cells with Bone Morphogenetic Protein 4 and Low Serum

Providing fully mature and functional osteoblasts is challenging for bone tissue engineering and regenerative medicine. Such cells could be obtained from multipotent bone marrow mesenchymal stem cells (MSCs) after induction by different osteogenic factors. However, there are some discrepancies in results, notably due to the use of sera and to the type of osteogenic factor. In this study, we compared the osteogenic differentiation of bone marrow MSCs induced by dexamethasone (Dex) or bone morphogenetic proteins (BMPs) by assessing phenotypes in vitro and functional osteoblasts in vivo. Reducing the content of fetal calf serum from 10% to 2% significantly increased the mineral deposition and expression of osteoblastic markers during osteogenesis. In comparison to Dex condition, the addition of BMP4 greatly improved the differentiation of MSCs into fully mature osteoblasts as seen by high expression of Osterix. These results were confirmed in different supportive matrixes, plastic flasks, or biphasic calcium phosphate biomaterials. In contrast to Dex-derived osteoblasts, BMP4-derived osteoblasts from MSCs were significantly able to produce new bone in subcutis of nude mice in accordance with in vitro results. In conclusion, we describe a convenient ex vivo method to produce consistently mature functional osteoblasts from human MSCs with use of BMP4 and low serum.

Establishment and characterization of the rhabdomyosarcoma cell line designated NUTOS derived from the human tongue sarcoma: Special reference to the susceptibility of anti-cancer drugs

Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.