[Multivariable analysis of tooth loss in subjects with severe periodontitis over 4-year natural progression].

OBJECTIVE: To evaluate the characteristics of severe periodontitis with various number of tooth loss during 4-year natural progression, and to analyze the factors related to higher rate of tooth loss. METHODS: A total of 217 patients aged 15 to 44 years with severe periodontitis were included, who participated in a 4-year natural progression research. Data obtained from questionnaire survey, clinical examination and radiographic measurement. Tooth loss during 4-year natural progression was evaluated. The baseline periodontal disease related and caries related factors were calculated, including number of teeth with bone loss > 50%, number of missing molars, number of teeth with widened periodontal ligament space (WPDL), number of teeth with periapical lesions and etc. Characteristics of populations with various number of tooth loss and the related factors that affected higher rate of tooth loss were analyzed. RESULTS: In 4 years of natural progression, 103 teeth were lost, and annual tooth loss per person was 0.12±0.38. Nine patients lost 3 or more teeth. Thirty-four patients lost 1 or 2 teeth, and 174 patients were absent of tooth loss. Molars were mostly frequent to lose, and canines presented a minimum loss. The number of teeth with WPDL, with periapical lesions, with intrabony defects, with probing depth (PD)≥7 mm, with PD≥5 mm, with clinical attachment loss≥5 mm, with bone loss > 50% and with bone loss > 65% were positively correlated to number of tooth loss. Results from orderly multivariate Logistic regression showd that the number of teeth with bone loss > 50% OR=1.550), baseline number of molars lost (OR=1.774), number of teeth with WPDL (1 to 2: OR=1.415; ≥3: OR=13.105), number of teeth with periapical lesions (1 to 2: OR=4.393; ≥3: OR=9.526) and number of teeth with caries/residual roots (OR=3.028) were significant risk factors related to higher likelihood of tooth loss and multiple tooth loss. CONCLUSION: In 4 years of natural progression, the number of teeth with bone loss > 50%, baseline number of missing molars, number of teeth with WPDL, baseline number of teeth with periapical lesions and number of teeth with caries/residual roots were significantly related to higher risk of tooth loss and multiple tooth loss among Chinese young and middle-aged patients with severe periodontitis in rural areas.

Rapid Production of Cell-Laden Microspheres Using a Flexible Microfluidic Encapsulation Platform.

This study establishes a novel microfluidic platform for rapid encapsulation of cells at high densities in photocrosslinkable microspherical hydrogels including poly(ethylene glycol)-diacrylate, poly(ethylene glycol)-fibrinogen, and gelatin methacrylate. Cell-laden hydrogel microspheres are advantageous for many applications from drug screening to regenerative medicine. Employing microfluidic systems is considered the most efficient method for scale-up production of uniform microspheres. However, existing platforms have been constrained by traditional microfabrication techniques for device fabrication, restricting microsphere diameter to below 200 µm and making iterative design changes time-consuming and costly. Using a new molding technique, the microfluidic device employs a modified T-junction design with readily adjustable channel sizes, enabling production of highly uniform microspheres with cell densities (10-60 million cells mL-1 ) and a wide range of diameters (300-1100 µm), which are critical for realizing downstream applications, through rapid photocrosslinking (≈1 s per microsphere). Multiple cell types are encapsulated at rates of up to 1 million cells per min, are evenly distributed throughout the microspheres, and maintain high viability and appropriate cellular activities in long-term culture. This microfluidic encapsulation platform is a valuable and readily adoptable tool for numerous applications, including supporting injectable cell therapy, bioreactor-based cell expansion and differentiation, and high throughput tissue sphere-based drug testing assays.

TNF-α differently regulates TRPV2 and TRPV4 channels in human dental pulp cells.

AIM: To investigate the influence of tumour necrosis factor (TNF)-α on transient receptor potential channel vanilloid subfamily type 2 (TRPV2) and TRPV4 channels in human dental pulp cells (HDPCs), and explore the potential downstream signalling pathway mediating this process. METHODOLOGY: Immunofluorescence staining and ratiometric calcium imaging were used to confirm the expression and activation of TRPV2 and TRPV4 channels. Different regulations of 1 and 10 ng mL-1 as well as short- and long-term TNF-α treatments to TRPV2 and TRPV4 response were examined by RT-qPCR, Western blot analysis, flow cytometry and ratiometric calcium imaging. Functions of TNF receptor (TNFR)1 and p38 MAPK signalling pathways in this process were also detected by respective inhibitors. Immunoelectron microscopy (IEM) was used to examine long-term effect of TNF-α on TRPV2 expression at the subcellular level. Data were analysed statistically with t-test, and one-way analysis of variance was used with the non-parametric Mann-Whitney and Kruskal-Wallis tests. The level of significance was set at P < 0.05. RESULTS: TRPV2 and TRPV4 channels were activated by respective agonists in HDPCs. Neither TRPV2 nor TRPV4 channels were upregulated by 1 ng mL-1 TNF-α (P > 0.05). TRPV2, but not TRPV4, was upregulated by 10 ng mL-1 TNF-α (P < 0.05). Both short- and long-term treatments with 10 ng mL-1 TNF-α significantly enhanced TRPV2 responses, whereas only short-term treatment of TNF-α increased TRPV4 response (P < 0.05). Moreover, the inhibitors of TNFR and p38 both significantly decreased the TNF-α-induced up-regulation of TRPV channels (P < 0.05). At the subcellular level, prolonged TNF-α treatment significantly increased the functional expression of the TRPV2 channel especially in the nucleus, endoplasmic reticulum and mitochondria. CONCLUSIONS: Low and high concentrations, as well as short- and long-term TNF-α treatments regulated the activity of TRPV2 and TRPV4 channels in HDPCs differently, and this effect might be mediated by TNFR1 and p38 MAPK signalling pathways. IEM was used to confirm that prolonged TNF-α treatment significantly increased the functional expression of the TRPV2 channel at a subcellular level.

Mechanical force-mediated pathological cartilage thinning is regulated by necroptosis and apoptosis.

OBJECTIVE: This study aimed to identify the mechanisms underlying mandibular chondrocyte cell death and cartilage thinning in response to mechanical force. MATERIAL AND METHODS: An in vivo model (compressive mechanical force) and an in vitro model (TNF-α+cycloheximide) were used to induce mandibular chondrocyte necroptosis. Hematoxylin and eosin staining and transmission electron microscopy were used to assess histological and subcellular changes in mandibular chondrocyte. Immunohistochemistry, western blotting, and real-time PCR were performed to evaluate changes in necroptotic protein markers. Cell activity, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were examined in vitro. RESULTS: The expression of RIP1, RIP3 and Caspase-8 in mandibular chondrocytes significantly increased after 4 days of compressive mechanical force. Furthermore, the inhibition of necroptosis by Necrostatin-1 (Nec-1) or the inhibition of apoptosis by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) partially restored mechanical force-mediated mandibular cartilage thinning and chondrocyte death. Moreover, a synergistic effect on cell death inhibition and mandibular cartilage thickness restoration were found when treated with Nec-1+Z-VAD. The results of the in vitro model were in line with the in vivo ones, indicating that the changes in MMP and ROS generation contributed to mandibular chondrocyte apoptosis and necroptosis. CONCLUSION: In addition to apoptosis, necroptosis also plays critical roles in pathological changes in mandibular cartilage after compressive mechanical force stimulation, implying RIP1, a master protein that mediates both necroptosis and apoptosis, as a potential therapeutic target in temporal mandibular osteoarthritis.

Rechargeable anticandidal denture material with sustained release in saliva.

OBJECTIVE: Candida-induced denture stomatitis is a common debilitating problem among denture wearers. Previously, we described the fabrication of a new denture material that released antifungal drugs when immersed in phosphate buffered saline. Here, we use more clinically relevant immersion conditions (human saliva; 37°C) and measure miconazole release and bioactivity. MATERIALS AND METHODS: Disks were prepared by grafting PNVP [poly(N-vinyl-2-pyrrolidinone)] onto PMMA [poly(methylmethacrylate)] using plasma initiation (PMMA-g-PNVP) and then loaded with miconazole. Drug-loaded disks were immersed in 10-100% human saliva (1-30 days). Miconazole release was measured and then tested for bioactivity vs miconazole-sensitive and miconazole-resistant Candida isolates. RESULTS: HPLC was used to quantify miconazole levels in saliva. Miconazole-loaded disks released antifungal drug for up to 30 days. Higher drug release was found with higher concentrations of saliva, and, interestingly, miconazole solubility was increased with higher saliva concentrations. The released miconazole retained its anticandidal activity. After immersion, the residual miconazole could be quenched and the disks recharged. Freshly recharged disks displayed the same release kinetics and bioactivity as the original disks. Quenched disks could also be charged with chlorhexidine that displayed anticandidal activity. CONCLUSIONS: These results suggest that PMMA-g-PNVP is a promising new denture material for long-term management of denture stomatitis.

Modified transcrestal sinus floor elevation with concomitant implant placement in edentulous posterior maxillae with residual bone height of 5 mm or less: a non-controlled prospective study.

The aim of this study was to describe a modified transcrestal sinus floor elevation (mTSFE) technique and to evaluate its clinical effectiveness and reliability when residual bone height is severely reduced. Forty-three maxillary edentulous patients who met the inclusion criteria were enrolled. All patients underwent the mTSFE technique; 66 dental implants were inserted simultaneously. Patient-reported outcomes were assessed 2 weeks after surgery. Prosthetic crowns were placed 6 months after surgery. Radiographic analyses and clinical analyses were conducted to assess the clinical effectiveness and feasibility of mTSFE during a follow-up period of 2-8 years. The mean vertical bone increase after surgery was 8.09 mm, and it decreased to 6.56 mm at 6 months after surgery. Two cases of membrane perforation occurred during surgery and one implant was lost in the third year after surgery; the survival rate at the implant level was 98.48%. No severe postoperative complication was reported and the subjective feeling of patients was acceptable. This mTSFE technique could simplify the operative procedure and might be helpful to reduce intraoperative trauma, as well as to alleviate postoperative discomfort.

Endothelial colony forming cell rolling and adhesion supported by peptide-grafted hydrogels.

The aim of this study was to investigate the ability of peptides and peptide combinations to support circulating endothelial colony forming cell (ECFC) rolling and adhesion under shear flow, informing biomaterial design in moving toward rapid cardiovascular device endothelialization. ECFCs have high proliferative capability and can differentiate into endothelial cells, making them a promising cell source for endothelialization. Both single peptides and peptide combinations designed to target integrins α4ß1 and α5ß1 were coupled to poly(ethylene glycol) hydrogels, and their performance was evaluated by monitoring velocity patterns during the ECFC rolling process, in addition to firm adhesion (capture). Tether percentage and velocity fluctuation, a parameter newly defined here, were found to be valuable in assessing cell rolling velocity patterns and when used in combination were able to predict cell capture. REDV-containing peptides binding integrin α4ß1 have been previously shown to reduce ECFC rolling velocity but not to support firm adhesion. This study finds that the performance of REDV-containing peptides in facilitating ECFC dynamic adhesion and capture can be improved by combination with α5ß1 integrin-binding peptides, which support ECFC static adhesion. Moreover, when similar in length, the peptide combinations may have synergistic effects in capturing ECFCs. With matching lengths, the peptide combinations including CRRETAWAC(cyclic)+REDV, P_RGDS+KSSP_REDV, and P_RGDS+P_REDV showed high values in both tether percentage and velocity fluctuation and improvement in ECFC capture compared to the single peptides at the shear rate of 20 s-1. These newly identified peptide combinations have the potential to be used as vascular device coatings to recruit ECFCs. STATEMENT OF SIGNIFICANCE: Restoration of functional endothelium following placement of stents and vascular grafts is critical for maintaining long-term patency. Endothelial colony forming cells (ECFCs) circulating in blood flow are a valuable cell source for rapid endothelialization. Here we identify and test novel peptides and peptide combinations that can potentially be used as coatings for vascular devices to support rolling and capture of ECFCs from flow. In addition to the widely used assessment of final ECFC adhesion, we also recorded the rolling process to quantitatively evaluate the interaction between ECFCs and the peptides, obtaining detailed performance of the peptides and gaining insight into effective capture molecule design. Peptide combinations targeting both integrin α4ß1 and integrin α5ß1 showed the highest percentages of ECFC capture.

Droplet Microfluidics-Based Fabrication of Monodisperse Poly(ethylene glycol)-Fibrinogen Breast Cancer Microspheres for Automated Drug Screening Applications.

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

Efficacy of mechanical bowel preparation with polyethylene glycol in prevention of postoperative complications in elective colorectal surgery: a meta-analysis.

PURPOSE: The aim of this study was to estimate efficacy of mechanical bowel preparation with polyethylene glycol (PEG) in prevention of postoperative complications in elective colorectal surgery. METHOD: A literature search of MEDLINE (PubMed), EMBASE, and the Cochrane Library was done to identify randomized controlled trials involving comparison of postoperative complications after mechanical bowel preparation with PEG (PEG group) and no preparation (control group). A meta-analysis was set up to distinguish overall difference between the two groups. RESULTS: A total of five randomized controlled trials was identified according to our inclusion criteria. The use of PEG for mechanical bowel preparation did not significantly reduce the rate of surgical site infection (SSI; odds ratio (OR) 95% confidence interval (CI), 1.39 (0.85-2.25); P = 0.19) including incisional SSI (OR 95% CI, 1.44 (0.88-2.33); P = 0.15), organ/space SSI (OR 95% CI, 1.10 (0.43-2.78); P = 0.49), anastomotic leak (OR 95% CI,1.78 (0.95-3.33; P = 0.07), mortality (OR 95% CI, 1.24 (0.37-4.14; P = 0.73), infectious complications (OR 95% CI, 1.14 (0.62-2.08); P = 0.67), and hospital stay (weighted mean difference 95% CI, 2.17 (-2.90-7.25); P = 0.40) except main complications (OR 95% CI, 1.76 (1.09-2.85); P = 0.02), of which the rate increased significantly in the PEG group. CONCLUSION: The use of mechanical bowel preparation with PEG does not significantly lower postoperative complications in elective colorectal surgery.

[Clinical application and evaluation of socket shield technique].

Objective: To further improve socket shield technique. The treatment outcome and the key related factors in Asian population are evaluated by measuring the root fragment and alveolar crest parameters in immediate implant placement cases with socket shield. Methods: A total of 18 patients, with 21 implants placed using socket shield technique were included in this retrospective study. Fourteen implants of 11 cases were recruited from West China Hospital of Stomatology, Sichuan University, 7 implants of 7 cases were recruited from Stomatological Hospital of Chongqing Medical University. The relationship between the thickness of the root fragment, the vertical height of the root fragment and the dimensional changes of buccal alveolar bone in 6 months was analyzed respectively. The pink-white esthetic scores were evaluated. The complications were recorded, analyzed and preventive measures were put forward. Results: There is a negative correlation between the vertical height of tooth fragment and the buccal crest height reduction (r=-0.458, P=0.037). There were negative correlations between the thickness of the tooth fragment with the vertical dimensional changes (r=-0.574, P=0.007) and horizontal dimensional changes (r=-0.619, P=0.003) of buccal alveolar bone. Three cases with internal exposure were recorded during the treatment. No severe complications were observed. Every case achieved a satisfying pink-white esthetic outcome according to the existing treatment protocols. Conclusions: Rigorous case screening, delicate surgical procedures, and maintaining adequate thickness of the root are the key to achieve a good esthetic outcome of implant treatment with socket shield technique.

LepR-Expressing Stem Cells Are Essential for Alveolar Bone Regeneration.

Stem cells play a critical role in bone regeneration. Multiple populations of skeletal stem cells have been identified in long bone, while their identity and functions in alveolar bone remain unclear. Here, we identified a quiescent leptin receptor-expressing (LepR+) cell population that contributed to intramembranous bone formation. Interestingly, these LepR+ cells became activated in response to tooth extraction and generated the majority of the newly formed bone in extraction sockets. In addition, genetic ablation of LepR+ cells attenuated extraction socket healing. The parabiosis experiments revealed that the LepR+ cells in the healing sockets were derived from resident tissue rather than peripheral blood circulation. Further studies on the mechanism suggested that these LepR+ cells were responsive to parathyroid hormone/parathyroid hormone 1 receptor (PTH/PTH1R) signaling. Collectively, we demonstrate that LepR+ cells, a postnatal skeletal stem cell population, are essential for alveolar bone regeneration of extraction sockets.

Thrombomodulin improves early outcomes after intraportal islet transplantation.

Primary islet nonfunction due to an instant blood mediated inflammatory reaction (IBMIR) leads to an increase in donor islet mass required to achieve euglycemia. In the presence of thrombin, thrombomodulin generates activated protein C (APC), which limits procoagulant and proinflammatory responses. In this study, we postulated that liposomal formulations of thrombomodulin (lipo-TM), due to its propensity for preferential uptake in the liver, would enhance intraportal engraftment of allogeneic islets by inhibiting the IBMIR. Diabetic C57BL/6J mice underwent intraportal transplantation with B10.BR murine islets. In the absence of treatment, conversion to euglycemia was observed among 29% of mice receiving 250 allo-islets. In contrast, a single infusion of lipo-TM led to euglycemia in 83% of recipients (p = 0.0019). Fibrin deposition (p < 0.0001), neutrophil infiltration (p < 0.0001), as well as expression TNF-alpha and IL-beta (p < 0.03) were significantly reduced. Significantly, thrombotic responses mediated by human islets in contact with human blood were also reduced by this approach. Lipo-TM improves the engraftment of allogeneic islets through a reduction in local thrombotic and inflammatory processes. As an enzyme-based pharmacotherapeutic, this strategy offers the potential for local generation of APC at the site of islet infusion, during the initial period of elevated thrombin production.

[Three-dimensional finite element analysis of four-implants supported mandibular overdentures using two different attachments].

Objective: To compare the biomechanical characteristics of four-implants mandibular overdentures supported by Locator attachment or bar-clip attachment under different mechanical loads using three-dimensional finite element analysis method. Methods: Two different models of four-implants supported mandibular overdentures using Locator attachment and bar-clip attachment (hereinafter called Locator model and bar-clip model) were established. Each model was subjected to five different mechanical loading conditions: 100 N vertical loading in central incisor (vertical load of incisor), 100 N vertical loading or oblique loading in canine (vertical or oblique loads of canines), 100 N vertical or oblique loading in mandibular first molar (vertical or oblique loads of mandibular first molar). The stress distributions in implants, peri-implant bone and mucosa were recorded under the above five conditions to evaluate the effects of different attachments on the biomechanical properties of implant-supported mandibular overdentures. Results: Regardless of loading conditions and types of attachments, the stress concentration in implants were located at the neck of implants, and the stress concentration in peri-implant bone was located in the cortical bone. The stress values in mucosa were always much smaller than those in implants and cortical bone. Regardless of loading positions (on canine or on mandibular first molar), the maximum stress at the bone interface around the implant under lateral loading was much higher than that under vertical loading. Under various loading conditions, the stress in implants and cortical bone of the Locator model (the highest von Mise stress value was respectively 79.5 and 22.3 MPa) were lower than that of bar-clip model (the highest von Mise stress value was 110.3 and 28.7 MPa respectively) while the maximum compressive stress in mucosa (0.198 MPa) in Locator model was slightly higher than that in the bar-clip model (0.137 MPa). Conclusions: In clinical practice, the lateral force applied to the implant-retained overdenture should be minimized to avoid complications caused by pathological loads. Under the same loading condition, the stress distributions in overdenture using Locator attachment are more dispersed, which is more conducive to long-term stability of implants.

Engineering of an elastic large muscular vessel wall with pulsatile stimulation in bioreactor.

Tissue engineering offers a new approach for the construction of vascular substitutes in vitro with proper mechanical properties. Although success has been made in the engineering of small blood vessels (<6mm in diameter), it remains a challenge to engineer large vessels (>6mm in diameter) due to their insufficient biomechanical property. In the current study, an elastic large vessel wall (6mm in diameter) was engineered by loading a polyglycolic acid (PGA) unwoven fiber scaffold seeded with smooth muscle cells (SMCs) on a vessel reactor designed with dynamic culture conditions. SMCs were isolated from canine carotid artery and expanded before seeding on a PGA fiber mesh. The cell-seeded PGA mesh was then loaded on a vessel reactor and subjected to pulsatile stimuli. Grossly, an elastic vessel wall was formed after 8 weeks of dynamic engineering. Histological examination showed well-orientated smooth muscle cells and collagenous fibers in the group with dynamic culture. In addition, the phenotype of SMCs was confirmed by positive staining of smooth muscle alpha-actin and calponin. On the contrary, disorganized smooth muscle cells and collagenous fibers were observed in the group under static culture without stimuli. Furthermore, the engineered vessels under dynamic culture exhibited significant improvements on biomechanical property over the one from static culture. Our results indicate that the approach developed in the current work is efficient for large vessel engineering. This approach may also be suitable for the engineering of other tissues with muscular tubular structure.

Influence of SiRNA targeting survivin on chemosensitivity of H460/cDDP lung cancer cells.

We investigated the difference in survivin expression between a multidrug-resistant lung cancer cell line (H460/cDDP) and its parental counterpart (H460) and the influence of siRNA targeting survivin on the chemosensitivity of H460/cDDP. SiRNA targeting survivin was transfected into H460/cDDP cells using a liposome approach. Survivin mRNA and protein expression were significantly higher in H460/cDDP than H460 cells. The median inhibitory concentrations (IC(50)s) for cisplatin and paclitaxol in vitro against H460/cDDP cells were significantly lower in cells treated with survivin-specific siRNA than in control cells. Apoptosis and cleaved caspase-3 expression were analysed using annexin V and Western blotting, respectively, and showed a significant increase in apoptosis after treatment with the chemotherapeutic agents plus specific siRNA. Specific siRNA sensitized H460/cDDP cells to both cisplatin and paclitaxol. Thus, survivin appears to participate in the multidrug resistance mechanism of H460/cDDP cells and siRNA targeting survivin has the potential to increase the sensitivity of drug-resistant cancer cells to anticancer drugs.

DLC coatings: effects of physical and chemical properties on biological response.

Recent trials on diamond-like carbon (DLC) coated medical devices have indicated promise for blood interfacing applications. The literature is sparse regarding structural and compositional effects of DLC on cellular response. An important goal in optimizing blood-interfacing implants is minimal macrophage attachment, and maximal albumin:fibrinogen adsorption ratio. DLC coatings deposited by PACVD and FAD, were analysed with respect to sp3 content (EELS), hydrogen content (ERDA), surface composition (XPS), surface roughness (AFM), surface energy, albumin:fibrinogen adsorption ratio, and macrophage viability and attachment. We found that increasing surface roughness and surface energy enhanced the macrophage viability and the albumin:fibrinogen adsorption ratio. We also found that the higher the hydrogen content for a-C:Hs deposited by PACVD, the lower the albumin:fibrinogen adsorption ratio, and macrophage attachment. This suggests that hydrogen content may be an important factor for influencing the biological response of DLC surfaces. Macrophage cells spread well on all DLC surfaces, and the surface results indicated the non-toxic nature of the surfaces on the cells at the time points tested.

PEG-fibrinogen hydrogels for three-dimensional breast cancer cell culture.

Tissue-engineered three-dimensional (3D) cancer models employing biomimetic hydrogels as cellular scaffolds provide contextual in vitro recapitulation of the native tumor microenvironment, thereby improving their relevance for use in cancer research. This study reports the use of poly(ethylene glycol)-fibrinogen (PF) as a suitable biosynthetic hydrogel for the 3D culture of three breast cancer cell lines: MCF7, SK-BR-3, and MDA-MB-231. Modification of the matrix characteristics of PF hydrogels was achieved by addition of excess poly(ethylene glycol) diacrylate, which resulted in differences in Young's moduli, degradation behavior, release kinetics, and ultrastructural variations in scaffold microarchitecture. Cancer cells were maintained in 3D culture with high viability within these hydrogels and resulted in cell-type dependent morphological changes over time. Cell proliferation and 3D morphology within the hydrogels were visualized through immunofluorescence staining. Finally, spatial heterogeneity of colony area within the hydrogels was quantified, with peripheral cells forming colonies of higher area compared to those in the interior regions. Overall, PF-based hydrogels facilitate 3D culture of breast cancer cells and investigation of cellular behavior in response to varying matrix characteristics. PF-based cancer models could be potentially used in future investigations of cancer biology and in anti-cancer drug-testing applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 236-252, 2017.

Encapsulation of Equine Endothelial Colony Forming Cells in Highly Uniform, Injectable Hydrogel Microspheres for Local Cell Delivery.

A common challenge in cell therapy is the inability to routinely maintain survival and localization of injected therapeutic cells. Delivering cells by direct injection increases the flexibility of clinical applications, but may cause low cell viability and retention rates due to the high shear forces in the needle and mechanical wash out. In this study, we encapsulated endothelial colony forming cells (ECFCs) in poly(ethylene glycol)-fibrinogen (PF) hydrogel microspheres using a custom-built microfluidic device; this system supports rapid encapsulation of high cell concentrations (10 million cells per mL) and resulting cell-laden microspheres are highly uniform in shape and size. The encapsulated ECFCs were shown to have >95% viability and continued to rapidly proliferate. Expression of cell markers (von Willebrand factor, CD105, and CD14), the ability to form tubules on basement membrane matrix, and the ability to take up low-density lipoprotein were similar between pre- and post-encapsulated cells. Viability of encapsulated ECFCs was maintained after shear through 18-23-gauge needles. Ex vivo and in vivo cell delivery studies were performed by encapsulating and injecting autologous equine ECFCs subcutaneously into distal limb full-thickness wounds of adult horses. Injected ECFCs were visualized by labeling with fluorescent nanodots before encapsulation. One week after injection, confocal microscopy analysis of biopsies of the leading edges of the wounds showed that the encapsulated ECFCs migrated into the surrounding host tissue indicating successful retention and survival of the delivered ECFCs. Rapid, scalable cell encapsulation into PF microspheres was demonstrated to be practical for use in large animal cell therapy and is a clinically relevant method to maintain cell retention and survival after local injection.

Evidence from CD spectra and melting temperatures for stable Hoogsteen-paired oligomer duplexes derived from DNA and hybrid triplexes.

The pyr*pur.pyr type of nucleic acid triplex has a purine strand that is Hoogsteen-paired with a parallel pyrimidine strand (pyr*pur pair) and that is Watson-Crick-paired with an antiparallel pyrimidine strand (pur.pyr pair). In most cases, the Watson-Crick pair is more stable than the Hoogsteen pair, although stable formation of DNA Hoogsteen-paired duplexes has been reported. Using oligomer triplexes of repeating d(AG)12 and d(CT)12 or r(CU)12 sequences that were 24 nt long, we found that hybrid RNA*DNA as well as DNA*DNA Hoogsteen-paired strands of triplexes can be more stable than the Watson-Crick-paired strands at low pH. The structures and relative stabilities of these duplexes and triplexes were evaluated by circular dichroism (CD) spectroscopy and UV absorption melting studies of triplexes as a function of pH. The CD contributions of Hoogsteen-paired RNA*DNA and DNA*DNA duplexes were found to dominate the CD spectra of the corresponding pyr*pur.pyr triplexes.

Ca2+-Dependent Endoplasmic Reticulum Stress Regulates Mechanical Stress-Mediated Cartilage Thinning.

Our previous study identified that endoplasmic reticulum stress (ERS) plays a critical role in chondrocyte apoptosis and mandibular cartilage thinning in response to compressive mechanical force, although the underlying mechanisms remain elusive. Because the endoplasmic reticulum (ER) is a primary site of intracellular Ca(2+) storage, we hypothesized that Ca(2+)-dependent ERS might be involved in mechanical stress-mediated mandibular cartilage thinning. In this study, we used in vitro and in vivo models to determine Ca(2+) concentrations, histological changes, subcellular changes, apoptosis, and the expression of ERS markers in mandibular cartilage and chondrocytes. The results showed that in chondrocytes, cytosolic Ca(2+) ([Ca(2+)]i) was dramatically increased by compressive mechanical force. Interestingly, the inhibition of Ca(2+) channels by ryanodine and 2-aminoethoxydiphenyl borate, inhibitors of ryanodine receptors and inositol trisphosphate receptors, respectively, partially rescued mechanical force-mediated mandibular cartilage thinning. Furthermore, chondrocyte apoptosis was also compromised by inhibiting the increase in [Ca(2+)]i that occurred in response to compressive mechanical force. Mechanistically, the ERS induced by compressive mechanical force was also repressed by [Ca(2+)]i inhibition, as demonstrated by a decrease in the expression of the ER stress markers 78 kDa glucose-regulated protein (GRP78) and 94 kDa glucose-regulated protein (GRP94) at both the mRNA and protein levels. Collectively, these data identified [Ca(2+)]i as a critical mediator of the pathological changes that occur in mandibular cartilage under compressive mechanical force and shed light on the treatment of mechanical stress-mediated cartilage degradation.