Buccal Fat Pad as a Potential Source of Stem Cells for Bone Regeneration:an in vitro Study in Static and Dynamic Culture

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized mass of adipose tissue that can be easily obtained via the oral cavity without injury to the external body surface. Another advantage of BFP over subcutaneous fat is that its size appears to be similar among different people, independent of body weight and fat distribution. However, limited studies have been conducted on the osteogenic capability of stem cells derived from BFP (BFPSCs). In this study, the BFPSCs were characterized for their osteogenic differentiation potential especially in contact with a synthetic scaffold in a perfusion bioreactor. The features of BFPSCs were compared with bone marrow-derived stem cells (BMSCs) as a well known cell source for bone tissue engineering. Comparing BFPSCs with BMSCs indicated similar morphology, but faster proliferation rate of BMSCs. Moreover, when properly induced for two weeks, BFPSCs resembled BMSCs in the production of bone-specific markers, such as alkaline phosphatase, collagen, bone morphogenic protein (BMP), Runx2, and osteocalcin. Both cell types attached nicely to the pores of a gelatin-coated β-Tricalcium phosphate scaffolds. More osteogenic differentiation potential was observed for both cells under dynamic culture in a perfusion bioreactor compared with static culture. The highest collagen content and BMP production were observed in BFPSCs cultured in the bioreactor for two weeks. These results define BFP as a new, rich, and accessible source of stem cells for tissue engineering purposes

COMPARISON OF VARIOUS MEASUREMENT TECHNIQUES FOR CHARACTERIZING THE HYDRODYNAMICS OF GAS-SOLID FLUIDIZED BEDS

There are various techniques to characterize the hydrodynamic of fluidized beds. Nowadays, sensor development is widely used to determine the hydrodynamic state of a fluidized bed to improve control and safety of the operation of such reactors. The objective of this study was to compare the advantages and disadvantages of the intrusive and novel non-intrusive techniques. The measurement techniques investigated in this work were vibration of the bed, acoustic emission and pressure. Extensive measurements were carried out at different superficial gas velocities with different particle sizes. Vibration and acoustic emissions were recorded at 25 KHz for 30 s using accelerometer and microphone, respectively. Pressure fluctuation signals were also collected at a sampling frequency of 400 Hz. These measurements were used for investigating the changes in the flow structure, specifically the flow regime transitions. The recorded signals were processed using wavelet analysis and statistical tools. It was shown that the variation of standard deviation, skewness and kurtosis of vibration signals against superficial gas velocity of the bed obey the same trend for different techniques. Results indicated that analyzing the vibration and acoustic signals can be considered as effective non intrusive techniques to characterize the hydrodynamics of gas-solid fluidized beds and in some cases they show better prediction of the hydrodynamic parameters

Towards Vascularized Tissue Blocks Using a Suspension Bioprinted Blood Vessel

In order for engineered tissue grafts and eventually organs to successfully integrate in a clinical setting, a functional vascular network is imperative. Without vasculature, the tissue constructs cannot receive nutrients essential for their survival, but also lack the stimuli that determine the tissue’s biophysical properties i.e. cell fate determination, cell to cell junctions, and cell orientation. In order for the vascular network to functionally connect to the patient, a hierarchical organization, resembling the vascular tree, is important. From previous studies it is known that fluid flow is a crucial component in controlling the formation of the vascular tree, and that the organization of the vascular network can be further controlled using gradients of angiogenic growth factors such as VEGF. By utilizing spheroid bioprinting within a microgel suspension, an artificial vessel structure was assembled. The deposited spheroids maintained viability and fused over time into perfusable vessels.The subsequent formation of small-diameter vascular structures and capillaries was regulated by an on-demand flow through the bioprinted vessel, resulting in controllable fluid flow shear stresses. Furthermore, VEGF was spatially patterned in the tissue block by locally doping the suspension with growth factor releasing microparticles. By varying both these stimuli, the location of vascular sprout formation and subsequent growth of the new vascular structures could be influenced. This spheroid 3D bioprinting platform offers a dynamic, customizable and accurate method to trigger and control the process of angiogenesis in vitro. By stimulating an artificial blood vessel with controlled fluid flow and growth factor gradients, a vascular complex vascular network can be produced and modulated. The combination of this approach with a gradual replacement of the microgel suspension with cells, can pave the way for the production of vascularized tissue blocks

Surface Coating of Polyurethane Films with Gelatin, Aspirin and Heparin to Increase the Hemocompatibility of Artificial Vascular Grafts

Purpose: A hemocompatible substrate can offer a wonderful facility for nitric oxide (NO) production by vascular endothelial cells in reaction to the inflammation following injuries. NO inhibits platelet aggregation this is especially critical in small-diameter vessels. Methods: The substrate films were made of polyurethane (PU) in a casting process and after plasma treatments, their surface was chemically decorated with polyethylene glycol (PEG) 2000, gelatin, gelatin-aspirin, gelatin-heparin and gelatin-aspirin-heparin. The concentrations of these ingredients were optimized in order to achieve the biocompatible values and the resulting modifications were characterized by water contact angle and Fourier transform infra-red (FTIR) assays. The values of NO production and platelet adhesion were then examined. Results: The water contact angle of the modified surface was reduced to 26±4⸰ and the newly developed hydrophilic chemical groups were confirmed by FTIR. The respective concentrations of 0.05 mg/ml and 100 mg/mL were found to be the IC50 values for aspirin and heparin. However, after the surface modification with aspirin, the bioactivity of the substrate increased in compared to the other experimental groups. In addition, there was a synergistic effect between these reagents for NO synthesis. While, heparin inhibited platelet adhesion more than aspirin. Conclusion: Because of the highly hydrophilic nature of heparin, this reagent was hydrolyzed faster than aspirin and therefore its influence on platelet aggregation and cell growth was greater. Taken together, the results give the biocompatible concentrations of both biomolecules that are required for endothelial cell proliferation, NO synthesis and platelet adhesion

Buccal fat pad as a potential source of stem cells for bone regeneration: an in vitro study

Adipose tissues hold great promise in bone tissue engineering since they are available in large quantities as a waste material. The buccal fat pad (BFP) is a specialized mass of adipose tissue that can be easily obtained via the oral cavity without injury to the external body surface. Another advantage of BFP over subcutaneous fat is that its size appears to be similar among different people, independent of body weight and fat distribution. However, limited studies have been conducted on the osteogenic capability of stem cells derived from BFP (BFPSCs). In this study, the BFPSCs were characterized for their osteogenic differentiation potential especially in contact with a synthetic scaffold in a perfusion bioreactor. The features of BFPSCs were compared with bone marrow-derived stem cells (BMSCs) as a well known cell source for bone tissue engineering. Comparing BFPSCs with BMSCs indicated similar morphology, but faster proliferation rate of BMSCs. Moreover, when properly induced for two weeks, BFPSCs resembled BMSCs in the production of bone-specific markers, such as alkaline phosphatase, collagen, bone morphogenic protein (BMP), Runx2, and osteocalcin. Both cell types attached nicely to the pores of a gelatin-coated β-Tricalcium phosphate scaffolds. More osteogenic differentiation potential was observed for both cells under dynamic culture in a perfusion bioreactor compared with static culture. The highest collagen content and BMP production were observed in BFPSCs cultured in the bioreactor for two weeks. These results define BFP as a new, rich, and accessible source of stem cells for tissue engineering purposes

Nitric oxide secretion by endothelial cells in response to fluid shear stress, aspirin, and temperature

Current vascular grafts have a high incidence of failure, especially in the grafts less than 6 mm in diameter, due to thrombus formation. Nitric oxide (NO) is released by endothelium and has some beneficial influences such as an antithrombotic effect. We hypothesized that applying different shear stress regiments and low temperature or aspirin would result in an increase in the amount of NO release from human umbilical vein endothelial cells (HUVECs) and decrease in platelet aggregation in the same manner as expected in vivo. HUVECs were cultured into the intraluminal surface of silicone tubes. HUVECs were subjected for 60 min to different parameters of shear stress, temperature, aspirin, and platelets or a combination in a perfusion bioreactor by monitoring NO secretion. We found that shear stress leads to an elevation of NO production in HUVECS, independent of the shear stress magnitude (0.9 or 1.8 dyne/cm2). The magnitude of this response increased with a decrease in temperature. Our results also show that by addition of platelets in combination with aspirin to media circulation, no thrombus formation occurred during the test time. Presence of aspirin resulted in marked increase in NO levels. In conclusion, shear stresses, temperature lowering, and aspirin increase the amount of NO release from HUVECs. Also no thrombus formation was detected in our experimental setting

Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues

Spatiotemporally controlled growth factor (GF) delivery is crucial for achieving functional vasculature within engineered tissues. However, conventional GF delivery systems show inability to recapitulate the dynamic and heterogeneous nature of developing tissue's biochemical microenvironment. Herein, an aptamer-based programmable GF delivery platform is described that harnesses dynamic affinity interactions for facilitating spatiotemporal control over vascular endothelial GF (VEGF165) bioavailability within gelatin methacryloyl matrices. The platform showcases localized VEGF165 sequestration from the culture medium (offering spatial-control) and leverages aptamer-complementary sequence (CS) hybridization for triggering VEGF165 release (offering temporal-control), without non-specific leakage. Furthermore, extensive 3D co-culture studies (human umbilical vein-derived endothelial cells & mesenchymal stromal cells), in bi-phasic hydrogel systems revealed its fundamentally novel capability to selectively guide cell responses and manipulate lumen-like microvascular networks via spatiotemporally controlling VEGF165 bioavailability within 3D microenvironment. This platform utilizes CS as an external biochemical trigger for guiding vascular morphogenesis which is suitable for creating dynamically controlled engineered tissues