The Uniform Distribution of Hydroxyapatite in a Polyurethane Foam-Based Scaffold (PU/HAp) to Enhance Bone Repair in a Calvarial Defect Model.

Polyurethane (PU) is a promising material for addressing challenges in bone grafting. This study was designed to enhance the bone grafting capabilities of PU by integrating hydroxyapatite (HAp), which is known for its osteoconductive and osteoinductive potential. Moreover, a uniform distribution of HAp in the porous structure of PU increased the effectiveness of bone grafts. PEG/APTES-modified scaffolds were prepared through self-foaming reactions. A uniform pore structure was generated during the spontaneous foaming reaction, and HAp was uniformly distributed in the PU structure (PU15HAp and PU30HAp) during foaming. Compared with the PU scaffolds, the HAp-modified PU scaffolds exhibited significantly greater protein absorption. Importantly, the effect of the HAp-modified PU scaffold on bone repair was tested in a rat calvarial defect model. The microstructure of the newly formed bone was analyzed with microcomputed tomography (µ-CT). Bone regeneration at the defect site was significantly greater in the HAp-modified PU scaffold group than in the PU group. This innovative HAp-modified PU scaffold improves current bone graft materials, providing a promising avenue for improved bone regeneration.

G Protein-Coupled Estrogen Receptor Mediates Cell Proliferation through the cAMP/PKA/CREB Pathway in Murine Bone Marrow Mesenchymal Stem Cells.

Estrogen is an important hormone to regulate skeletal physiology via estrogen receptors. The traditional estrogen receptors are ascribed to two nuclear estrogen receptors (ERs), ERα and ERß. Moreover, G protein-coupled estrogen receptor-1 (GPER-1) was reported as a membrane receptor for estrogen in recent years. However, whether GPER-1 regulated osteogenic cell biology on skeletal system is still unclear. GPER-1 is expressed in growth plate abundantly before puberty but decreased abruptly since the very late stage of puberty in humans. It indicates GPER-1 might play an important role in skeletal growth regulation. GPER-1 expression has been confirmed in osteoblasts, osteocytes and chondrocytes, but its expression in mesenchymal stem cells (MSCs) has not been confirmed. In this study, we hypothesized that GPER-1 is expressed in bone MSCs (BMSC) and enhances BMSC proliferation. The cultured tibiae of neonatal rat and murine BMSCs were tested in our study. GPER-1-specific agonist (G-1) and antagonist (G-15), and GPER-1 siRNA (siGPER-1) were used to evaluate the downstream signaling pathway and cell proliferation. Our results revealed BrdU-positive cell counts were higher in cultured tibiae in the G-1 group. The G-1 also enhanced the cell viability and proliferation, whereas G-15 and siGPER-1 reduced these activities. The cAMP and phosphorylation of CREB were enhanced by G-1 but inhibited by G-15. We further demonstrated that GPER-1 mediates BMSC proliferation via the cAMP/PKA/p-CREB pathway and subsequently upregulates cell cycle regulators, cyclin D1/cyclin-dependent kinase (CDK) 6 and cyclin E1/CDK2 complex. The present study is the first to report that GPER-1 mediates BMSC proliferation. This finding indicates that GPER-1 mediated signaling positively regulates BMSC proliferation and may provide novel insights into addressing estrogen-mediated bone development.

G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice.

Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown-rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

In Vivo Longitudinal Tracking of Lymphangiogenesis and Angiogenesis in Cutaneous Melanoma Mouse Model Using Multifunctional Optical Coherence Tomography.

Melanoma is a high-risk skin cancer because it tends to metastasize early and ultimately leads to death. In this study, we introduced a noninvasive multifunctional optical coherence tomography (MFOCT) for the early detection of premetastatic pathogenesis in cutaneous melanoma by label-free imaging of microstructures (i.e., providing the thickness and the scattering information) and microcirculation (i.e., providing depth-resolved angiography and lymphangiography). Using MFOCT-based approaches, we presented an in vivo longitudinal observation of the tumor microenvironment in Braf V600E/V600E ;Pten -/- mice with inducible melanoma monitored for 42 days. Quantitative analysis of MFOCT images identified an increased number of lymphatic and vascular vessels during tumor progression and faster lymphangiogenesis (beginning on day 21) than angiogenesis (beginning on day 28) in the melanoma microenvironment. We further observed lymphatic vessel enlargement from the first week of melanoma development, implying tumor cells interacting with the vessels and increased likelihood of metastasis. MFOCT identified cutaneous melanoma‒associated angiogenesis and lymphangiogenesis before the possible visual perception of the tumor (≥42 days) and before metastasis could be diagnosed using micropositron emission tomography (35 days). Thus, the proposed quantitative analysis using MFOCT has the potential for early detection of cutaneous melanoma progression or prediction of metastatic melanoma in a mouse model. However, retrospective and extensive experiments still need to be performed in the future to confirm the value of MFOCT in clinical application.

Inhibition of melanin synthesis and melanosome transfer by chitosan biomaterials.

Decreasing skin pigmentation is desirable for various medical or cosmetic conditions. Although numerous pharmaceutical agents are currently available, their depigmentation effects are still not satisfactory. In this study, we investigated the effects of chitosan, a natural marine product, on melanin synthesis and melanosome transfer. Treating B16F10 melanoma cells caused the inhibitory effect of chitosan on melanogenesis to be more prominent under α-melanocyte-stimulating hormone (α-MSH) stimulation. Chitosan samples of different molecular weights inhibited melanogenesis to a comparable extent, whereas increasing the deacetylation of chitosan enhanced its depigmentation effects. Chitosan was found to effectively reduce basal or α-MSH-stimulated melanogenesis by suppressing the expression of melanogenic-related proteins (microphthalmia transcription factor, tyrosinase, and tyrosinase-related protein-1 and protein-2) as well as inhibiting tyrosinase activity. Moreover, the inhibitory effect of chitosan on melanogenesis in human melanocytes was confirmed. A transwell coculture system using permeable inserts was designed to allow the contact of human melanocytes and human HaCaT keratinocytes through the tiny holes on the membrane. When chitosan was added to this melanocyte-keratinocyte coculture system, we observed decreased melanosome release from melanocytes. Reduced melanosome uptake by keratinocytes was also observed, and was probably mediated by inhibiting protease-activated receptor 2 expression. Many skin-whitening agents can modulate the process of melanogenesis, but few have been shown to inhibit the melanosome transfer and uptake process. We demonstrated that chitosan exhibits a robust effect on depigmentation by inhibiting melanogenesis as well as melanosome transfer and uptake. Therefore, chitosan represents a potential therapeutic agent for hyperpigmentation disorders.

Proton pump inhibitor use and risk of hip fracture in patients with type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is associated with a high rate of comorbidity, including osteoporosis and peptic ulcers. Proton pump inhibitors (PPIs) are a group of acid-suppressing drugs commonly used for treating peptic ulcers. However, observational studies have reported an association between PPI therapy and osteoporotic fractures. This study investigated the association between PPI use and hip fracture (HFx) among patients with T2DM. We conducted this population-based propensity-matched retrospective cohort study using the National Health Insurance Research Database in Taiwan. Patients newly diagnosed with T2DM between 2000 and 2008 were identified. After excluding those who previously used PPIs or suffered HFx, 398,885 patients were recruited (44,341 PPI users; 354,544 non-users). HFx risk data from 2000 to 2013 were collected to calculate the cumulative rate of HFx in these two groups. Sensitivity analyses were conducted to evaluate the effects of PPI dose. After propensity score matching of 1:4, 44,431 and 177,364 patients were assigned to the PPI user and non-user groups, respectively. PPI user group showed an increased risk of HFx with an adjusted hazard ratio of 1.41 (95% CI 1.29-1.54) without dose-response relationship. Thus, there is an increased risk of HFx in patients with T2DM receiving long-term PPI treatment.

Effects of fibroblasts on the function of acinar cells from the same human parotid gland.

BACKGROUND: Artificial salivary gland replacement would be an ideal treatment for xerostomia. In vivo, salivary gland cells are surrounded by a complex stromal environment in which fibroblasts are the main cell type in proximity to the gland cells. However, very little is known about the relationship between these fibroblasts and the gland cells. METHODS: Parotid gland acinar cells (PGACs) and fibroblasts from the same human gland were cocultured. PGAC function-related protein expression was investigated. RESULTS: The expression of α-amylase in PGACs was increased in a fibroblast ratio-dependent manner. Both fibroblast-conditioned medium and direct coculture also significantly enhanced the PGAC expression of α-amylase. Basic fibroblast growth factor (bFGF) seems to be a regulator of α-amylase expression in PGACs. CONCLUSION: An appropriate number of fibroblasts in contact with the PGACs is necessary to promote PGAC function. Fibroblast-secreted bFGF may play a paracrine signaling role in the regulation of α-amylase expression in PGACs. © 2015 Wiley Periodicals, Inc. Head Neck 38: E279-E286, 2016.

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and ßIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. STATEMENT OF SIGNIFICANCE: To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and ßIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts.

Increased mucociliary differentiation and aquaporins formation of respiratory epithelial cells on retinoic acid-loaded hyaluronan-derivative membranes.

While playing a major role in maintaining the mucociliary phenotype of respiratory epithelial cells (RECs), retinoids are critical determinants of their normal function. However, despite being a powerful biological agent, retinoic acid (RA) is generally not used in regenerative medicine due to its scarce bioavailability via conventional administration. Therefore, the ability to incorporate RA into biomaterials allows for a combination of the biological effects of RA and biomaterials in influencing cellular behavior. This study attempts to develop RA-loaded hyaluronan-derivative membrane (RA-HAm) and investigates how this membrane affects the mucociliary differentiation and aquaporins (AQP) formation of RECs. In a simulated in vitro culture condition, the RA release from membranes is maintained for 7days. On the seventh day, the cumulative release rate of RA from supportive biomaterials is ~87% under detect limitation. RECs cultured on RA-HAm reveal numerous mature ciliated cells and microvilli compared to aggregated cilia-like structures on hyaluronan-derivative membrane (HAm). Moreover, the expression levels of MUC5AC and AQP on RA-HAm are higher than those on HAm. The proposed model elucidates the release of hydrophobic RA from hyaluronan-derivative biomaterials. We believe that RA-loaded hyaluronan biomaterials are highly promising biomaterials for use in sinonasal surgery and tissue engineering of the respiratory system.

Photo-induced antitumor effect of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide.

We have applied a fluorescent molecule 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) for tumor targeting and treatment. In this study, we investigated the photo-induced antitumor effect of BMVC. In vitro cell line studies showed that BMVC significantly killed TC-1 tumor cells at light dose greater than 40 J/cm(2). The fluorescence of BMVC in the tumor peaked at 3 hours and then gradually decreased to reach the control level after 24 hours. In vivo tumor treatment studies showed BMVC plus light irradiation (iPDT) significantly inhibited the tumor growth. At day 24 after tumor implantation, tumor volume was measured to be 225 ± 79 mm(3), 2542 ± 181 mm(3), 1533 ± 766 mm(3), and 1317 ± 108 mm(3) in the iPDT, control, light-only, and BMVC-only groups, respectively. Immunohistochemistry studies showed the microvascular density was significantly lower in the iPDT group. Taken together, our results demonstrated that BMVC may be a potent tumor-specific photosensitizer (PS) for PDT.

Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin.

As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.

Early reperfusion improves the recovery of contralateral electrophysiological diaschisis following focal cerebral ischemia in rats.

OBJECTIVES: We evaluated electrophysiological benefits of reperfusion following ischemic stroke. METHODS: Rats received either transient proximal occlusion of the right middle cerebral artery for 30 (Group I, n=8) or 90 minutes (Group II, n=8) or permanent thermocoagulation of the distal right middle cerebral artery (Group III, n=6). Neurobehavioral outcome and somatosensory evoked potentials (SSEPs) were examined before and at 7 days after the onset of brain ischemia. Brain infarction was assessed after the rats were euthanized. RESULTS: Before ischemia, stable SSEPs were consistently recorded. At 7 days post-insult, Group III (permanent occlusion) had the greatest reduction in the SSEPs recorded ipsilaterally and contralaterally. Groups I and II (transient ischemic groups) also had depressant SSEPs recorded from the ipsilateral ischemic and the contralateral intact brain (electrophysiological diaschisis). However, prolonged ischemia resulted in greater brain infarction and increased neurological deficits in addition to greater reductions in the ipsilateral and the contralateral SSEPs. CONCLUSION: Early reperfusion facilitates the electrophysiological recovery in both ipsilateral lesional and the contralateral intact brain, which may be closely relevant to post-injury brain rewiring. We also demonstrated that contralateral electrophysiological diaschisis could be greatly reversed by early reperfusion and is independent of the lesion size of striatum.