Novel Artificial Scaffold for Bone Marrow Regeneration: Honeycomb Tricalcium Phosphate.

Bone marrow is complex structure containing heterogenetic cells, making it difficult to regenerate using artificial scaffolds. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which is a cylindrical scaffold with a honeycomb arrangement of straight pores, and we demonstrated that TCP with 300 and 500 µm pore diameters (300TCP and 500TCP) induced bone marrow structure within the pores. In this study, we examined the optimal scaffold structure for bone marrow with homeostatic bone metabolism using honeycomb TCP. 300TCP and 500TCP were transplanted into rat muscle, and bone marrow formation was histologically assessed. Immunohistochemistry for CD45, CD34, Runt-related transcription factor 2 (Runx2), c-kit single staining, Runx2/N-cadherin, and c-kit/Tie-2 double staining was performed. The area of bone marrow structure, which includes CD45(+) round-shaped hematopoietic cells and CD34(+) sinusoidal vessels, was larger in 300TCP than in 500TCP. Additionally, Runx2(+) osteoblasts and c-kit(+) hematopoietic stem cells were observed on the surface of bone tissue formed within TCP. Among Runx2(+) osteoblasts, spindle-shaped N-cadherin(+) cells existed in association with c-kit(+)Tie-2(+) hematopoietic stem cells on the bone tissue formed within TCP, which formed a hematopoietic stem cell niche similar to as in vivo. Therefore, honeycomb TCP with 300 µm pore diameters may be an artificial scaffold with an optimal geometric structure as a scaffold for bone marrow formation.

SOD3 Expression in Tumor Stroma Provides the Tumor Vessel Maturity in Oral Squamous Cell Carcinoma.

Tumor angiogenesis is one of the hallmarks of solid tumor development. The progressive tumor cells produce the angiogenic factors and promote tumor angiogenesis. However, how the tumor stromal cells influence tumor vascularization is still unclear. In the present study, we evaluated the effects of oral squamous cell carcinoma (OSCC) stromal cells on tumor vascularization. The tumor stromal cells were isolated from two OSCC patients with different subtypes: low invasive verrucous squamous carcinoma (VSCC) and highly invasive squamous cell carcinoma (SCC) and co-xenografted with the human OSCC cell line (HSC-2) on nude mice. In comparison, the CD34+ vessels in HSC-2+VSCC were larger than in HSC-2+SCC. Interestingly, the vessels in the HSC-2+VSCC expressed vascular endothelial cadherin (VE-cadherin), indicating well-formed vascularization. Our microarray data revealed that the expression of extracellular superoxide dismutase, SOD3 mRNA is higher in VSCC stromal cells than in SCC stromal cells. Moreover, we observed that SOD3 colocalized with VE-cadherin on endothelial cells of low invasive stroma xenograft. These data suggested that SOD3 expression in stromal cells may potentially regulate tumor vascularization in OSCC. Thus, our study suggests the potential interest in SOD3-related vascular integrity for a better OSCC therapeutic strategy.

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model.

The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue-derived stromal cells (G-SCs and P-SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate-resistant acid phosphatase staining in a cell line-derived xenograft model. The results demonstrated that G-SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC-3 cell line, whereas P-SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC-3 cells. Furthermore, the effects of G-SCs, P-SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)-9, membrane type 1 MMP (MT1-MMP), Snail, parathyroid hormone-related peptide (PTHrP) and receptor activator of NF-κB ligand (RANKL) in HSC-3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G-SCs had a more prominent effect on the expression of MMP-9, MT1-MMP, Snail, PTHrP, and RANKL, whereas P-SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP-9, MT1-MMP and Snail expression. The potential genes underlying the differential effects of G-SCs and P-SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin-dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G-SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P-SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC.

Crosstalk between cancer and different cancer stroma subtypes promotes the infiltration of tumor­associated macrophages into the tumor microenvironment of oral squamous cell carcinoma.

Tumor­associated macrophages (TAMs) are linked to the progression of numerous types of cancer. However, the effects of the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC), particularly the cancer stroma on TAMs, remains to be elucidated. In the present study, the effects of verrucous SCC­associated stromal cells (VSCC­SCs), SCC­associated stromal cells (SCC­SCs) and human dermal fibroblasts (HDFs) on the differentiation, proliferation and migration of macrophages in vitro was assayed using Giemsa staining, and immunofluorescence, MTS and Transwell (migration) assays, respectively. The combined results suggested that both VSCC­SCs and SCC­SCs promoted the differentiation of macrophages into M2 type TAMs, as well as the proliferation and migration of macrophages following crosstalk with HSC­3 cells in vitro. Moreover, the SCC­SCs exerted a more prominent effect on TAMs than the VSCC­SCs. Immunohistochemical staining was used to examine the expression of CD34, CD45, CD11b and CD163 to assay the effects of VSCC­SCs, SCC­SCs and HDFs on microvessel density (MVD) and the infiltration of CD45(+) monocytes, CD11b(+) TAMs and CD163(+) M2 type macrophages. The results suggested that both VSCC­SCs and SCC­SCs promoted MVD and the infiltration of CD45(+) monocytes, CD11b(+) TAMs and CD163(+) M2 type TAMs into the TME of OSCC following crosstalk with HSC­3 cells in vivo. The SCC­SCs exerted a more prominent promoting effect than the VSCC­SCs. Finally, the potential genes underlying the differential effects of VSCC­SCs and SCC­SCs on the infiltration of TAMs were investigated using microarray analysis. The results revealed that interleukin 1ß, bone morphogenetic protein 4, interleukin 6 and C­X­C motif chemokine ligand 12 had great potential to mediate the differential effects of VSCC­SCs and SCC­SCs on TAM infiltration. On the whole, the findings presented herein, demonstrate that both VSCC­SCs and SCC­SCs promote the infiltration of TAMs into the TME of OSCC following crosstalk with HSC­3 cells; the SCC­SCs were found to exert a more prominent promoting effect. This may represent a potential regulatory mechanism for the infiltration of TAMs into the TME of OSCC.

Significance of cancer stroma for bone destruction in oral squamous cell carcinoma using different cancer stroma subtypes.

Stromal cells in the tumor microenvironment (TME) can regulate the progression of numerous types of cancer; however, the bone invasion of oral squamous cell carcinoma (OSCC) has been poorly investigated. In the present study, the effect of verrucous SCC­associated stromal cells (VSCC­SCs), SCC­associated stromal cells (SCC­SCs) and human dermal fibroblasts on bone resorption and the activation of HSC­3 osteoclasts in vivo were examined by hematoxylin and eosin, AE1/3 (pan­cytokeratin) and tartrate­resistant acid phosphatase staining. In addition, the expression levels of matrix metalloproteinase (MMP)9, membrane­type 1 MMP (MT1­MMP), Snail, receptor activator of NF­κB ligand (RANKL) and parathyroid hormone­related peptide (PTHrP) in the bone invasion regions of HSC­3 cells were examined by immunohistochemistry. The results suggested that both SCC­SCs and VSCC­SCs promoted bone resorption, the activation of osteoclasts, and the expression levels of MMP9, MT1­MMP, Snail, RANKL and PTHrP. However, SCC­SCs had a more prominent effect compared with VSCC­SCs. Finally, microarray data were used to predict potential genes underlying the differential effects of VSCC­SCs and SCC­SCs on bone invasion in OSCC. The results revealed that IL1B, ICAM1, FOS, CXCL12, INS and NGF may underlie these differential effects. In conclusion, both VSCC­SCs and SCC­SCs may promote bone invasion in OSCC by enhancing the expression levels of RANKL in cancer and stromal cells mediated by PTHrP; however, SCC­SCs had a more prominent effect. These findings may represent a potential regulatory mechanism underlying the bone invasion of OSCC.

Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment.

Accumulating evidence has shown that cancer stroma and BM-derived cells (BMDCs) in the tumor microenvironment (TME) play vital roles in tumor progression. However, the mechanism by which oral cancer stroma recruits any particular subset of BMDCs remains largely unknown. Here, we sought to identify the subset of BMDCs that is recruited by cancer stroma. We established a sequential transplantation model in BALB/c nude mice, including (a) BM transplantation of GFP-expressing cells and (b) coxenografting of patient-derived stroma (PDS; 2 cases, designated PDS1 and PDS2) with oral cancer cells (HSC-2). As controls, xenografting was performed with HSC-2 alone or in combination with normal human dermal fibroblasts (HDF). PDS1, PDS2, and HDF all promoted BMDC migration in vitro and recruitment in vivo. Multicolor immunofluorescence revealed that the PDS coxenografts recruited Arginase-1+CD11b+GR1+GFP+ cells, which are myeloid-derived suppressor cells (MDSCs), to the TME, whereas the HDF coxenograft did not. Screening using microarrays revealed that PDS1 and PDS2 expressed CCL2 mRNA (encoding C-C motif chemokine ligand 2) at higher levels than did HDF. Indeed, PDS xenografts contained significantly higher proportions of CCL2+ stromal cells and CCR2+Arginase-1+CD11b+GR1+ MDSCs (as receiver cells) than the HDF coxenograft. Consistently, a CCL2 synthesis inhibitor and a CCR2 antagonist significantly inhibited the PDS-driven migration of BM cells in vitro. Furthermore, i.p. injection of the CCR2 antagonist to the PDS xenograft models significantly reduced the CCR2+Arginase-1+CD11b+GR1+ MDSC infiltration to the TME. In conclusion, oral cancer stroma-secreted CCL2 is a key signal for recruiting CCR2+ MDSCs from BM to the TME.

Stromal cells in the tumor microenvironment promote the progression of oral squamous cell carcinoma.

The stromal cells in the tumor microenvironment (TME) can influence the progression of multiple types of cancer; however, data on oral squamous cell carcinoma (OSCC) are limited. In the present study, the effects of verrucous squamous cell carcinoma­associated stromal cells (VSCC­SCs), squamous cell carcinoma­associated stromal cells (SCC­SCs) and human dermal fibroblasts (HDFs) on the tumor nest formation, proliferation, invasion and migration of HSC­3 cells were examined in vitro using Giemsa staining, MTS, and Transwell (invasion and migration) assays, respectively. The results revealed that both the VSCC­SCs and SCC­SCs inhibited the tumor nest formation, and promoted the proliferation, invasion and migration of OSCC cells in vitro. Furthermore, the effects of VSCC­SCs, SCC­SCs and HDFs on the differentiation, proliferation, invasion and migration of OSCC cells in vivo were evaluated by hematoxylin and eosin staining, tartrate­resistant acid phosphatase staining, immunohistochemistry and double­fluorescent immunohistochemical staining, respectively. The results demonstrated that the VSCC­SCs promoted the differentiation, proliferation, invasion and migration of OSCC cells, while the SCC­SCs inhibited the differentiation, and promoted the proliferation, invasion and migration of OSCC cells in vivo. Finally, microarray data were used to predict genes in VSCC­SCs and SCC­SCs that may influence the progression of OSCC, and those with potential to influence the differential effects of VSCC­SCs and SCC­SCs on the differentiation of OSCC. It was found that C­X­C motif chemokine ligand (CXCL)8, mitogen­activated protein kinase 3 (MAPK3), phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit alpha (PIK3CA), C-X-C motif chemokine ligand 1 (CXCL1) and C­C motif chemokine ligand 2 (CCL2) may be involved in the crosstalk between VSCC­SCs, SCC­SCs and OSCC cells, which regulates the progression of OSCC. Intercellular adhesion molecule 1 (ICAM1), interleukin (IL)1B, Fos proto­oncogene, AP­1 transcription factor subunit (FOS), bone morphogenetic protein 4 (BMP4), insulin (INS) and nerve growth factor (NGF) may be responsible for the differential effects of VSCC­SCs and SCC­SCs on the differentiation of OSCC. On the whole, the present study demonstrates that both VSCC­SCs and SCC­SCs may promote the progression of OSCC, and SCC­SCs were found to exert a more prominent promoting effect; this may represent a potential regulatory mechanism for the progression of OSCC.

The Origin of Stroma Influences the Biological Characteristics of Oral Squamous Cell Carcinoma.

Normal stromal cells surrounding the tumor parenchyma, such as the extracellular matrix (ECM), normal fibroblasts, mesenchymal stromal cells, and osteoblasts, play a significant role in the progression of cancers. However, the role of gingival and periodontal ligament tissue-derived stromal cells in OSCC progression is unclear. In this study, the effect of G-SCs and P-SCs on the differentiation, proliferation, invasion, and migration of OSCC cells in vitro was examined by Giemsa staining, Immunofluorescence (IF), (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), invasion, and migration assays. Furthermore, the effect of G-SCs and P-SCs on the differentiation, proliferation, and bone invasion by OSCC cells in vivo was examined by hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Finally, microarray data and bioinformatics analyses identified potential genes that caused the different effects of G-SCs and P-SCs on OSCC progression. The results showed that both G-SCs and P-SCs inhibited the differentiation and promoted the proliferation, invasion, and migration of OSCC in vitro and in vivo. In addition, genes, including CDK1, BUB1B, TOP2A, DLGAP5, BUB1, and CCNB2, are probably involved in causing the different effects of G-SCs and P-SCs on OSCC progression. Therefore, as a potential regulatory mechanism, both G-SCs and P-SCs can promote OSCC progression.

Cancer-Associated Stromal Cells Promote the Contribution of MMP2-Positive Bone Marrow-Derived Cells to Oral Squamous Cell Carcinoma Invasion.

Tumor stromal components contribute to tumor development and invasion. However, the role of stromal cells in the contribution of bone marrow-derived cells (BMDCs) in oral squamous cell carcinoma (OSCC) invasion is unclear. In the present study, we created two different invasive OSCC patient-derived stroma xenografts (PDSXs) and analyzed and compared the effects of stromal cells on the relation of BMDCs and tumor invasion. We isolated stromal cells from two OSCC patients: less invasive verrucous OSCC (VSCC) and highly invasive conventional OSCC (SCC) and co-xenografted with the OSCC cell line (HSC-2) on green fluorescent protein (GFP)-positive bone marrow (BM) cells transplanted mice. We traced the GFP-positive BM cells by immunohistochemistry (IHC) and detected matrix metalloproteinase 2 (MMP2) expression on BM cells by double fluorescent IHC. The results indicated that the SCC-PDSX promotes MMP2-positive BMDCs recruitment to the invasive front line of the tumor. Furthermore, microarray analysis revealed that the expressions of interleukin 6; IL-6 mRNA and interleukin 1 beta; IL1B mRNA were higher in SCC stromal cells than in VSCC stromal cells. Thus, our study first reports that IL-6 and IL1B might be the potential stromal factors promoting the contribution of MMP2-positive BMDCs to OSCC invasion.

High glucose inhibits proliferation and differentiation of osteoblast in alveolar bone by inducing pyroptosis.

The inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone are well documented. However, a comprehensive study focused on the molecular mechanisms is still unknown. Recent studies have revealed that caspase-1 participates in the pathological processes of hepatic injury, cancers and diabetes related complications. However, the relationship between pyroptosis and proliferation and differentiation of osteoblasts has not been investigated. This study aimed to explore the possible pyroptosis participating in the inhibition of high glucose on the proliferation and differentiation of osteoblast in alveolar bone. The diabetes model was constructed both in vitro and in vivo to detect the expression of pyroptosis related factors. These results show that high glucose inhibits proliferation and differentiation of osteoblast in alveolar bone through pyroptosis pathway. Furthermore, caspase-1 inhibitor was co-administered with high glucose in ME3T3-E1 cells, which shows that caspase-1 inhibitor could repress effect of high glucose on the proliferation and differentiation of osteoblast. In conclusion, High glucose could activate the pyroptosis through the caspase-1/GSDMD/IL-1ß pathway to inhibit the proliferation and differentiation of osteoblast in alveolar bone, which provides a theoretical basis for clinical treatment of alveolar bone disease in diabetic patients.

Anthocyanin is involved in the activation of pyroptosis in oral squamous cell carcinoma.

BACKGROUND: The anti-carcinogenic effects of anthocyanin are well documented. Oral squamous cell carcinoma is one of the most common and lethal cancer types due to its high degree of malignancy and poor prognosis. The main purpose of the current study was to investigate the potential inhibitory effects of anthocyanin on oral squamous cell carcinoma and identify effective targets for therapy. METHODS: Cell viability was measured using cell counting kit-8 (CCK8). Cell migration and invasion abilities were determined using scratch-wound and Transwell invasion assays, respectively. mRNA and protein expression patterns of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3), caspase-1 and IL-1ß were detected using qRT-PCR, immunofluorescence and western blot. The gasdermin D (GSDMD) level was determined via confocal microscopy and western blot. RESULTS: Anthocyanin reduced the viability of oral squamous cell carcinoma cells and inhibited migration and invasion abilities. Simultaneously, activation of pyroptosis was associated with enhanced expression of NLRP3, caspase-1, and IL-1ß. Upon administration of caspase-1 inhibitors, anthocyanin-activated pyroptosis was suppressed and cell viability, migration, and invasion rates concomitantly enhanced. CONCLUSION: Anthocyanin promotes the death of oral squamous cell carcinoma cells through activation of pyroptosis and inhibits tumor progression.