Prognosticating the effect of temperature and pH parameters on size and stability of the nanoliposome system based on thermodynamic modeling.

The main challenge of using nanoliposome systems is controlling their size and stability. In order to overcome this challenge, according to the research conducted at the Research Centre for New Technologies of Biological Engineering, University of Tehran, a model for predicting the size and stability of nanoliposome systems based on thermodynamic relations has been presented. In this model, by using the presented equations and without performing many experiments in the laboratory environment, the effect of temperature, ionic power and different pH can be considered simultaneously whereas examining the components of size, stability and any feature were considered before. Synthesis and application of liposomal nanocarriers in different operating conditions can be investigated and predicted, and due to the change in temperature and pH, the smallest size of th system can be obtained. In this study, we were able to model the synthesis and storage conditions of liposomal nanocarriers at different temperatures and acidic, neutral and alkaline pHs, based on the calculation of mathematical equations. This model also indicates that with increasing temperature, the radius increases but with increasing pH, the radius first increases and then decreases. Therefore, this model can be used to predict size and stability in different operating conditions. In fact, with this modelling method, there is no need to study through laboratory methods and analysis to determine the size, stability and surface loads, and in terms of Accuracy, time and cost savings are affordable.

Stability improvement of polyaniline nanocomposite immunosensor for early detection of insulin receptor antibody as biomarker of type 2 diabetes.

Polyaniline nanocomposite with controllable properties was used to design and fabricate a novel electrochemical immunosensor for the early detection of type 2 diabetes. Insulin receptor antibody is a powerful predictor of type 2 diabetes development in individuals. A systematic study was carried out to investigate the effects of different polyaniline layers and the Nafion layer on the morphological, chemical, and electrochemical properties of nanocomposite immunosensor, especially the stability. The bioengineered Nafion-Au nanoparticles-polyaniline/gold electrode demonstrated outstanding electrocatalytic performance in the detection of insulin receptor antibodies with a high sensitivity (136.21 µA.ng-1.ml.cm-2) in a linear range from 0.001 to 200 ng.ml-1 as well as a low detection limit of 1.827 pg.ml-1, response time within 10 min, remarkable selectivity, and significant stability of 80 days. Therefore, the developed immunosensor is a suitable nanocomposite platform for insulin receptor antibody level determination in human plasma.

Advances in bioreactors for lung bioengineering: From scalable cell culture to tissue growth monitoring.

Lung bioengineering has emerged to resolve the current lung transplantation limitations and risks, including the shortage of donor organs and the high rejection rate of transplanted lungs. One of the most critical elements of lung bioengineering is bioreactors. Bioreactors with different applications have been developed in the last decade for lung bioengineering approaches, aiming to produce functional reproducible tissue constructs. Here, the current status and advances made in the development and application of bioreactors for bioengineering lungs are comprehensively reviewed. First, bioreactor design criteria are explained, followed by a discussion on using bioreactors as a culture system for scalable expansion and proliferation of lung cells, such as producing epithelial cells from induced pluripotent stem cells (iPSCs). Next, bioreactor systems facilitating and improving decellularization and recellularization of lung tissues are discussed, highlighting the studies that developed bioreactors for producing engineered human-sized lungs. Then, monitoring bioreactors are reviewed, showing their ability to evaluate and optimize the culture conditions for maturing engineered lung tissues, followed by an explanation on the ability of ex vivo lung perfusion systems for reconditioning the lungs before transplantation. After that, lung cancer studies simplified by bioreactors are discussed, showing the potentials of bioreactors in lung disease modeling. Finally, other platforms with the potential of facilitating lung bioengineering are described, including the in vivo bioreactors and lung-on-a-chip models. In the end, concluding remarks and future directions are put forward to accelerate lung bioengineering using bioreactors.

Flow Preconditioning of Endothelial Cells on Collagen-Immobilized Silicone Fibers Enhances Cell Retention and Antithrombotic Function.

Stability and antithrombotic functionality of endothelial cells on silicone hollow fibers (SiHFs) are critical in the development of biohybrid artificial lungs. Here we aimed to enhance endothelial cell retention and anti-thrombotic function by low (12 dyn/cm2 , 24 h) fluid shear stress ("flow") preconditioning of endothelial cells seeded on collagen-immobilized SiHFs. The response of endothelial cells without preconditioning (48 h static culture) and with preconditioning (24 h static culture followed by 24 h flow preconditioning) on hollow fibers to high fluid shear stress (30 dyn/cm2 , 1 h) was assessed in a parallel-plate flow chamber. Finite element (FE) modeling was used to simulate shear stress within the flow chamber. We found that collagen immobilization on hollow fibers using carbodiimide bonds provided sufficient stability to high shear stress. Flow preconditioning for 24 h before treatment with high shear stress for 1 h on collagen-immobilized hollow fibers increased cell retention (1.3-fold). The FE model showed that cell flattening due to flow preconditioning reduced maximum shear stress on cells by 32%. Flow preconditioning prior to exposure to high fluid shear stress enhanced the production of nitric oxide (1.3-fold) and prostaglandin I2 (1.2-fold). In conclusion, flow preconditioning of endothelial cells on collagen-immobilized SiHFs enhanced cell retention and antithrombotic function, which could significantly improve current biohybrid artificial lungs.

Preparation, characterization, and silanization of 3D microporous PDMS structure with properly sized pores for endothelial cell culture.

Nowadays, application of porous polydimethylsiloxane (PDMS) structure in biomedical is becoming widespread, and many methods have been established to create such structure. Although the pores created through these methods are mostly developed on the outer surface of PDMS membrane, this study offers a simple and cost-efficient technique for creating three-dimensional (3D) microporous PDMS structure with appropriate pore size for endothelial cell culture. In this study, combination of gas foaming and particulate leaching methods, with NaHCO3 as effervescent salt and NaCl as progen are used to form a 3D PDMS sponge. The in situ chemical reaction between NaHCO3 and HCl resulted in the formation of small pores and channels. Moreover, soaking the samples in HCl solution temporarily improved the hydrophilicity of PDMS, which then facilitated the penetration of water for further leaching of NaCl. The surface chemical modification process was performed by (3-aminopropyl)triethoxysilane to culture endothelial cells on porous PDMS matrix. The results are an indication of positive response of endothelial cells to the fabricated PDMS sponge. Because of simplicity and practicality of this method for preparing PDMS sponge with appropriate pore size and biological properties, the fabricated matrix can perfectly be applied to future studies in blood-contacting devices.

Use of sulfur-oxidizing bacteria as recognition elements in hydrogen sulfide biosensing system.

Four sulfur-oxidizing bacteria (Thiobacillus thioparus, Acidithiobacillus thiooxidans PTCC1717, Acidithiobacillus ferrooxidans PTCC1646, and Acidithiobacillus ferrooxidans PTCC1647) were used as biorecognition elements in a hydrogen sulfide biosensing system. All the experiments were performed in 0.1 M phosphate buffer solution containing 1-20 ppm H2S with optimum pH and temperature for each species. Although H2 S was applied to the biosensing system, the dissolved O2 content decreased. Dissolved O2 consumed by cells in both free and immobilized forms was measured using a dissolved oxygen sensor. Free bacterial cells exhibit fast response (<200 Sec). Immobilization of the cells on polyvinyl alcohol was optimized using an analytical software. Immobilized A. ferrooxidans and A. thiooxidans retained more than 50% of activity after 30 days of immobilization. According to the data, A. thiooxidans and A. ferrooxidans are appropriate species for hydrogen sulfide biosensor.

Applying shear stress to endothelial cells in a new perfusion chamber: hydrodynamic analysis.

Perfusion bioreactors have been proved to be an impartible part of vascular tissue engineering due to its broad range of applications as a means to distribute nutrients within porous scaffold along with providing appropriate physical and mechanical stimuli. To better understand the mechanical phenomena inside a bioreactor, computational fluid dynamics (CFD) was adopted followed by a validation technique. The fluid dynamics of the media inside the bioreactor was modeled using the Navier-Stokes equation for incompressible fluids while convection through the scaffold was described by Brinkman's extension of Darcy's law for porous media. Flow within the reactor determined the orientation of endothelial cells on the scaffold. To validate flow patterns, streamlines and shear stresses, colorimetry technique was used following attained results from CFD. Our bioreactor was modeled to simulate the optimum condition and flow patterns over scaffold to culture ECs for in vitro experimentation. In such experiments, cells were attached firmly without significant detachment and more noticeably elongation process was triggered even shortly after start up.

Osteogenic Activity on NaOH-Etched Three-Dimensional-Printed Poly-ɛ-Caprolactone Scaffolds in Perfusion or Spinner Flask Bioreactor.

Bioreactor systems, for example, spinner flask and perfusion bioreactors, and cell-seeded three-dimensional (3D)-printed scaffolds are used in bone tissue engineering strategies to stimulate cells and produce bone tissue suitable for implantation into the patient. The construction of functional and clinically relevant bone graft using cell-seeded 3D-printed scaffolds within bioreactor systems is still a challenge. Bioreactor parameters, for example, fluid shear stress and nutrient transport, will crucially affect cell function on 3D-printed scaffolds. Therefore, fluid shear stress induced by spinner flask and perfusion bioreactors might differentially affect osteogenic responsiveness of pre-osteoblasts inside 3D-printed scaffolds. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine fluid shear stress and osteogenic responsiveness of MC3T3-E1 pre-osteoblasts seeded on the scaffolds in the bioreactors using finite element (FE)-modeling and experiments. FE-modeling was used to quantify wall shear stress (WSS) distribution and magnitude inside 3D-printed PCL scaffolds within spinner flask and perfusion bioreactors. MC3T3-E1 pre-osteoblasts were seeded on NaOH surface-modified 3D-printed PCL scaffolds, and cultured in customized static, spinner flask, and perfusion bioreactors up to 7 days. The scaffolds' physicochemical properties and pre-osteoblast function were assessed experimentally. FE-modeling showed that spinner flask and perfusion bioreactors locally affected WSS distribution and magnitude inside the scaffolds. The WSS distribution was more homogeneous inside scaffolds in perfusion than in spinner flask bioreactors. The average WSS on scaffold-strand surfaces ranged from 0 to 6.5 mPa for spinner flask bioreactors, and from 0 to 4.1 mPa for perfusion bioreactors. Surface modification of scaffolds by NaOH resulted in a surface with a honeycomb-like pattern and increased surface roughness (1.6-fold), but decreased water contact angle (0.3-fold). Both spinner flask and perfusion bioreactors increased cell spreading, proliferation, and distribution throughout the scaffolds. Perfusion, but not spinner flask bioreactors more strongly enhanced collagen (2.2-fold) and calcium deposition (2.1-fold) throughout the scaffolds after 7 days compared with static bioreactors, likely due to uniform WSS-induced mechanical stimulation of the cells revealed by FE-modeling. In conclusion, our findings indicate the importance of using accurate FE models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems. Impact Statement The success of cell-seeded three-dimensional (3D)-printed scaffolds depends on cell stimulation by biomechanical/biochemical factors to produce bone tissue suitable for implantation into the patient. We designed and fabricated surface-modified 3D-printed poly-ɛ-caprolactone (PCL) scaffolds, as well as static, spinner flask, and perfusion bioreactors to determine wall shear stress (WSS) and osteogenic responsiveness of pre-osteoblasts seeded on the scaffolds using finite element (FE)-modeling and experiments. We found that cell-seeded 3D-printed PCL scaffolds within perfusion bioreactors more strongly enhanced osteogenic activity than within spinner flask bioreactors. Our results indicate the importance of using accurate FE-models to estimate WSS and determine experimental conditions for designing cell-seeded 3D-printed scaffolds in bioreactor systems.

A theoretical model for evaluation of the design of a hollow-fiber membrane oxygenator.

Geometric data are fundamental to the design of a contactor. The efficiency of a membrane contactor is mainly defined by its mass-transfer coefficient. However, design modifications also have significant effects on the performance of membrane contactors. In a hollow-fiber membrane oxygenator (HFMO), properties such as priming volume and effective membrane surface area (referred to as design specifications) can be determined. In this study, an extensive theoretical model for calculation of geometric data and configuration properties, and, consequently, optimization of the design of an HFMO, is presented. Calculations were performed for Oxyphan(®) hollow-fiber micro-porous membranes, which are frequently used in current HFMOs because of their high gas exchange performance. The results reveal how to regulate both the transverse and longitudinal pitches of fiber bundles to obtain a lower rand width and a greater number of windings. Such modifications assist optimization of module design and, consequently, substantially increase the efficiency of an HFMO. On the basis of these considerations, three values, called efficiency factors, are proposed for evaluation of the design specifications of an HFMO with regard with its performance characteristics (i.e. oxygen-transfer rate and blood pressure drop). Moreover, the performance characteristics of six different commercial HFMOs were measured experimentally, in vitro, under the same standard conditions. Comparison of calculated efficiency factors reveals Quadrox(®) is the oxygenator with the most efficient design with regard with its performance among the oxygenators tested.

Ultrasensitive early detection of insulin antibody employing novel electrochemical nano-biosensor based on controllable electro-fabrication process.

An ultrasensitive novel electrochemical nano-biosensor for rapid detection of insulin antibodies against diabetes antigens was developed in this research. The presence of insulin antibodies has been demonstrated to be a strong predictor for the development of type 1 diabetes in individuals who do not have diabetes but are genetically predisposed. The proposed nano-biosensor fabrication process was based on the optimized sequential electropolymerization of polyaniline and electrodeposition of gold nanoparticles on the surface of the functionalized gold electrode. The morphological and chemical characterization of the modified electrode was studied by field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and micro Raman spectroscopy. Moreover, the role of each component in the modification of the electrode was studied by electrochemical methods systematically. After immobilizing insulin antigen and blocking with bovine serum albumin, the nano-biosensor was used for determining different concentrations of insulin antibody under the optimal conditions. This nano-biosensor could respond to insulin antibody with a linear calibration range from 0.001 ng ml-1 to 1000 ng ml-1 with the detection limit of 0.017 pg ml-1 and 0.034 pg ml-1 and selectivity of 18.544 µA ng-1 ml.cm-2 and 31.808 µA ng-1 ml.cm-2 via differential pulse voltammetry and square wave voltammetry, respectively. This novel nano-biosensor exhibited a short response time, high sensitivity, and good reproducibility. It was successfully used in determining the insulin antibody in human samples with a standard error of less than 0.178. Therefore, the nano-biosensor has the potential for the application of early detection of type 1 diabetes. To our best knowledge, label-free electrochemical detection of insulin antibody based on immunosensor is developed for the first time.

Decellularized human-sized pulmonary scaffolds for lung tissue engineering: a comprehensive review.

The ultimate goal of lung bioengineering is to produce transplantable lungs for human beings. Therefore, large-scale studies are of high importance. In this paper, we review the investigations on decellularization and recellularization of human-sized lung scaffolds. First, studies that introduce new ways to enhance the decellularization of large-scale lungs are reviewed, followed by the investigations on the xenogeneic sources of lung scaffolds. Then, decellularization and recellularization of diseased lung scaffolds are discussed to assess their usefulness for tissue engineering applications. Next, the use of stem cells in recellularizing acellular lung scaffolds is reviewed, followed by the case studies on the transplantation of bioengineered lungs. Finally, the remaining challenges are discussed, and future directions are highlighted.

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration.

Background: One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using three-dimensional printing (3DP). Herein, we aimed to determine whether the much tighter control of microstructure of 3DP poly(lactic-co-glycolic) acid/ß-tricalcium phosphate (PLGA/ß-TCP) scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods: Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results: TThe 3DP scaffolds had a smoother surface (Ra: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (Ra: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. Alkaline phosphatase (ALP) activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion: The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/ß-TCP scaffolds are possibly more favorable for bone formation.

Enhanced articular cartilage decellularization using a novel perfusion-based bioreactor method.

Current decellularization methods for articular cartilages require many steps, various and high amounts of detergents, and a relatively long time to produce decellularized scaffolds. In addition, such methods often damage the essential components and the structure of the tissue. This study aims to introduce a novel perfusion-based bioreactor (PBB) method to decellularize bovine articular cartilages efficiently while reducing the harmful physical and chemical steps as well as the duration of the process. This leads to better preservation of the structure and the essential components of the native tissue. Firstly, a certain number of channels (Ø 180 µm) were introduced into both sides of cylindrical articular bovine cartilage disks (5 mm in diameter and 1 mm in thickness). Next, the disks were decellularized in the PBB and a shaker as the control. Using the PBB method resulted in ∼90% reduction of DNA content in the specimens, which was significantly higher than those of the shaker results with ∼60%. Also, ∼50% sulfated glycosaminoglycan (sGAG) content and ∼92% of the compression properties were maintained implying the efficient preservation of the structure and components of the scaffolds. Moreover, the current study indicated that the PBB specimens supported the adherence and proliferation of the new cells effectively. In conclusion, the results show that the use of PBB method increases the efficiency of producing decellularized cartilage scaffolds with a better maintenance of essential components and structure, while reducing the chemicals and steps required for the process. This will pave the way for producing close-to-natural scaffolds for cartilage tissue engineering.

Cross-linked acellular lung for application in tissue engineering: Effects on biocompatibility, mechanical properties and immunological responses.

The concept of providing tissue engineering scaffolds with natural physical properties and minimal immunogenicity has not been systematically approached for the lungs yet. Here, the rat acellular lung tissue (ALT) was cross-linked to provide either EDC/NHS cross-linked tissue (EDC/NHS-CLT) or tannic acid cross-linked tissue (TA-CLT). Young's modulus revealed that EDC/NHS-CLT had mechanical properties similar to the native lung and culture of lung mesenchymal cells showed a higher potential of cell proliferation on EDC/NHS-CLT versus TA-CLT and ALT. The in vitro immunogenicity tests showed a strong induction of T-cell proliferation by TA-CLT and an attenuated macrophage induction by TA-CLT. Processed rat lungs were implanted xenogenically into the mouse peritoneal cavity and the host-implant interactions showed that tannic acid is not released from TA-CLT in a physiologically effective dose. The profile of peritoneal fluid proinflammatory (TNFα, IL-1ß, IL-12p70 and IL-17) and anti-inflammatory (IL-10 and TGFß1) cytokines, and CD3+ T-lymphocytes and CD11b+ macrophages revealed that apart from induction of high levels of IL-17 during the first week and IL-10 during the second to third weeks after implantation by TA-CLT, other indicators of immune reactions to cross-linked tissues were not significantly different from ALT. Also, a high fibrotic reaction to TA-CLT was observed on the weeks 2-3, but alveolar structures were preserved in EDC/NHS-CLT. Our findings show that by controlled EDC/NHS cross-linking, an acellular lung scaffold could be provided with mechanical properties similar to native lung, which promotes mesenchymal lung cells proliferation and does not stimulate recipient's immune system more than a non-cross-linked tissue.

Determination of CO2 sensitivity of micro-organisms in shaken bioreactors. I. Novel method based on the resistance of sterile closure.

Influence of carbon dioxide on growth and product kinetics of industrially important micro-organisms is essential for the interpretation of a bioprocess. In this research, the CO2 effects on productivity and growth rate of micro-organisms have been studied by using a variety of kplug. The applied method is based on a different concentration of CO2 in the headspace of ventilation flasks. The presented method is simple, inexpensive and shows similar results compared to large-scale fermentation regarding the evolution of CO2 in a batch system. For the investigation of the proposed method, experiments employing Arxula adeninivorans LS3, Corynebacterium glutamicum (DM1730 and ATCC WT13032) and Hansenula polymorpha DSM70277 as model organisms in the ventilation flasks are performed. The fermentations in the RAMOS (respiratory activity monitoring system) device were carried out with a normal aeration rate (1 vvm) and under the same operating conditions as the ventilation flask f1. The modified unsteady-state model was used to predict the operation conditions of a biological system in the ventilation flasks. In the present study, a novel and easy method for the quantification of CO2 sensitivity of micro-organisms in shaken bioreactors (called ventilation flask) was achieved.

Determination of CO2 sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method.

In the present study, a new online monitoring method for the determination of the CO2 sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO2 sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO2 concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.

Role of biomechanics in vascularization of tissue-engineered bones.

Biomaterial based reconstruction is still the most commonly employed method of small bone defect reconstruction. Bone tissue-engineered techniques are improving, and adjuncts such as vascularization technologies allow re-evaluation of traditional reconstructive methods for healingofcritical-sized bone defect. Slow infiltration rate of vasculogenesis after cell-seeded scaffold implantation limits the use of clinically relevant large-sized scaffolds. Hence, in vitro vascularization within the tissue-engineered bone before implantation is required to overcome the serious challenge of low cell survival rate after implantation which affects bone tissue regeneration and osseointegration. Mechanobiological interactions between cells and microvascular mechanics regulate biological processes regarding cell behavior. In addition, load-bearing scaffolds demand mechanical stability properties after vascularization to have adequate strength while implanted. With the advent of bioreactors, vascularization has been greatly improved by biomechanical regulation of stem cell differentiation through fluid-induced shear stress and synergizing osteogenic and angiogenic differentiation in multispecies coculture cells. The benefits of vascularization are clear: avoidance of mass transfer limitation and oxygen deprivation, a significant decrease in cell necrosis, and consequently bone development, regeneration and remodeling. Here, we discuss specific techniques to avoid pitfalls and optimize vascularization results of tissue-engineered bone. Cell source, scaffold modifications and bioreactor design, and technique specifics all play a critical role in this new, and rapidly growing method for bone defect reconstruction. Given the crucial importance of long-term survival of vascular network in physiological function of 3D engineered-bone constructs, greater knowledge of vascularization approaches may lead to the development of new strategies towards stabilization of formed vascular structure.

3D-printed poly(Ɛ-caprolactone) scaffold with gradient mechanical properties according to force distribution in the mandible for mandibular bone tissue engineering.

In bone tissue engineering, prediction of forces induced to the native bone during normal functioning is important in the design, fabrication, and integration of a scaffold with the host. The aim of this study was to customize the mechanical properties of a layer-by-layer 3D-printed poly(ϵ-caprolactone) (PCL) scaffold estimated by finite element (FE) modeling in order to match the requirements of the defect, to prevent mechanical failure, and ensure optimal integration with the surrounding tissue. Forces and torques induced on the mandibular symphysis during jaw opening and closing were predicted by FE modeling. Based on the predicted forces, homogeneous-structured PCL scaffolds with 3 different void sizes (0.3, 0.6, and 0.9 mm) were designed and 3D-printed using an extrusion based 3D-bioprinter. In addition, 2 gradient-structured scaffolds were designed and 3D-printed. The first gradient scaffold contained 2 regions (0.3 mm and 0.6 mm void size in the upper and lower half, respectively), whereas the second gradient scaffold contained 3 regions (void sizes of 0.3, 0.6, and 0.9 mm in the upper, middle and lower third, respectively). Scaffolds were tested for their compressive and tensile strength in the upper and lower halves. The actual void size of the homogeneous scaffolds with designed void size of 0.3, 0.6, and 0.9 mm was 0.20, 0.59, and 0.95 mm, respectively. FE modeling showed that during opening and closing of the jaw, the highest force induced on the symphysis was a compressive force in the transverse direction. The compressive force was induced throughout the symphyseal line and reduced from top (362.5 N, compressive force) to bottom (107.5 N, tensile force) of the symphysis. Compressive and tensile strength of homogeneous scaffolds decreased by 1.4-fold to 3-fold with increasing scaffold void size. Both gradient scaffolds had higher compressive strength in the upper half (2 region-gradient scaffold: 4.9 MPa; 3 region-gradient scaffold: 4.1 MPa) compared with the lower half (2 region-gradient scaffold: 2.5 MPa; 3 region-gradient scaffold: 2.7 MPa) of the scaffold. 3D-printed PCL scaffolds had higher compressive strength in the scaffold layer-by-layer building direction compared with the side direction, and a very low tensile strength in the scaffold layer-by-layer building direction. Fluid shear stress and fluid pressure distribution in the gradient scaffolds were more homogeneous than in the 0.3 mm void size scaffold and similar to the 0.6 mm and 0.9 mm void size scaffolds. In conclusion, these data show that the mechanical properties of 3D-printed PCL scaffolds can be tailored based on the predicted forces on the mandibular symphysis. These 3D-printed PCL scaffolds had different mechanical properties in scaffold building direction compared with the side direction, which should be taken into account when placing the scaffold in the defect site. Our findings might have implications for improved performance and integration of scaffolds with native tissue.

Short Pretreatment with Calcitriol Is Far Superior to Continuous Treatment in Stimulating Proliferation and Osteogenic Differentiation of Human Adipose Stem Cells.

OBJECTIVE: This study investigated whether short stimulation (30 minutes) of human adipose stem cells (hASCs) with 1,25-dihydroxyvitamin D3 (calcitriol or 1,25-(OH)2VitD3), fitting within the surgical procedure time frame, suffices to induce osteogenic differentiation, and compared this with continuous treatment with 1,25-(OH)2VitD3. MATERIALS AND METHODS: In this experimental study, hASCs were pretreated with/without 10 nM calcitriol for 30 minutes, seeded on biphasic calcium phosphate (BCP), and cultured for 3 weeks with/without 1,25-(OH)2VitD3. Cell attachment was determined 30 minutes after cell seeding. AlamarBlue assay, alkaline phosphatase (ALP) assay, ALP staining, real-time polymerase chain reaction (PCR), and protein assay were used to evaluate the effect of short calcitriol pretreatment on proliferation and osteogenic differentiation of hASCs up to 3 weeks. RESULTS: Pretreatment with 1,25-(OH)2VitD3 enhanced the attachment of hASCs to BCP by 1.5-fold compared to nontreated cells and increased the proliferation by 3.5-fold at day 14, and 2.6-fold at day 21. In contrast, continuous treatment increased the proliferation by 1.7-fold only at day 14. After 2 weeks, ALP activity was increased by 18.5-fold when hASCs were pretreated with 1,25-(OH)2VitD3 for 30 minutes but increased only 2.6-fold when compared with its continuous counterpart. Moreover, after 14 days, pretreatment resulted in significant upregulation of the osteogenic markers RUNX2 and SPARC by 3.6-fold and 2.2-fold, respectively, while this was not observed upon continuous treatment. Finally, 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3 increased VEGF189 expression, which may contribute to the process of angiogenesis. CONCLUSION: This study is the first research showing that 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3, not only enhanced cell attachment to the scaffold at seeding time, but also promoted the proliferation and osteogenic differentiation of hASCs more strongly than continuous treatment, suggesting that short pre-treatment with 1,25-(OH)2VitD3 is a promising approach for the regeneration of bones in a one-step surgical procedure.

Inlet flow rate of perfusion bioreactors affects fluid flow dynamics, but not oxygen concentration in 3D-printed scaffolds for bone tissue engineering: Computational analysis and experimental validation.

Fluid flow dynamics and oxygen-concentration in 3D-printed scaffolds within perfusion bioreactors are sensitive to controllable bioreactor parameters such as inlet flow rate. Here we aimed to determine fluid flow dynamics, oxygen-concentration, and cell proliferation and distribution in 3D-printed scaffolds as a result of different inlet flow rates of perfusion bioreactors using experiments and finite element modeling. Pre-osteoblasts were treated with 1 h pulsating fluid flow with low (0.8 Pa; PFFlow) or high peak shear stress (6.5 Pa; PFFhigh), and nitric oxide (NO) production was measured to validate shear stress sensitivity. Computational analysis was performed to determine fluid flow between 3D-scaffold-strands at three inlet flow rates (0.02, 0.1, 0.5 ml/min) during 5 days. MC3T3-E1 pre-osteoblast proliferation, matrix production, and oxygen-consumption in response to fluid flow in 3D-printed scaffolds inside a perfusion bioreactor were experimentally assessed. PFFhigh more strongly stimulated NO production by pre-osteoblasts than PFFlow. 3D-simulation demonstrated that dependent on inlet flow rate, fluid velocity reached a maximum (50-1200 µm/s) between scaffold-strands, and fluid shear stress (0.5-4 mPa) and wall shear stress (0.5-20 mPa) on scaffold-strands surfaces. At all inlet flow rates, gauge fluid pressure and oxygen-concentration were similar. The simulated cell proliferation and distribution, and oxygen-concentration data were in good agreement with the experimental results. In conclusion, varying a perfusion bioreactor's inlet flow rate locally affects fluid velocity, fluid shear stress, and wall shear stress inside 3D-printed scaffolds, but not gauge fluid pressure, and oxygen-concentration, which seems crucial for optimized bone tissue engineering strategies using bioreactors, scaffolds, and cells.