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.
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•Jaw opening induced higher compression to upper versus lower parts of the symphysis.•All scaffolds had higher compressive strength in building versus side direction.•Gradient scaffolds had higher compressive strength in upper versus lower half.•Gradient scaffolds fit the force distribution in the symphysis during jaw opening.•Fluid shear stress and pressure were rather homogeneous in the gradient scaffolds.
Abstract Strontium Ranelate (SrRan) is used to decrease the risk of bone fractures. Any factor that alters the release of paracrine signals by osteocytes in response to mechanical stimuli potentially ...affects bone mass and structure, and thus fracture resistance. We hypothesized that SrRan affects paracrine signaling from mechanically-stimulated osteocytes towards osteoclast-precursors and osteoblasts. MLO-Y4 osteocytes were cultured for 24 h with SrRan (0.1–3 mM) and either or not mechanically stimulated by pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) for 60 min. Nitric oxide (NO) and prostaglandin E2 (PGE2 ) release, and expression of mechanoresponsive genes were quantified. Conditioned medium (CM) from osteocytes was added to mouse bone marrow cells for 7 days to assess osteoclastogenesis, or MC3T3-E1 osteoblasts for 4–16 days to measure osteogenic gene expression. SrRan (3 mM) enhanced NO and PGE2 release to the same extent in static osteocytes (NO: 1.6-fold; PGE2 : 2.8-fold) and PFF-stimulated osteocytes (NO: 1.3-fold; PGE2 : 2.6-fold). CM from PFF-treated osteocytes without SrRan enhanced Ki67 expression but reduced Runx2 and Ocn expression in osteoblasts. This effect on gene expression was not observed with CM obtained from osteocytes treated with the combination of PFF and 3 mM SrRan. CM from PFF-treated osteocytes inhibited osteoclastogenesis by 1.9-fold. The combination of PFF and 3 mM SrRan reduced osteocyte-stimulated osteoclastogenesis even more strongly (4.3-fold). In conclusion, SrRan affects paracrine signaling between mechanically-stimulated MLO-Y4 osteocytes and both osteoblasts and osteoclast precursors. The positive effects of SrRan on bone fracture resistance may thus be partly explained by altered paracrine signaling by osteocytes.
The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoforms TRalpha (1) TRalpha (2), TRbeta (1), and TRbeta (2) were investigated using in situ hybridization and ...immunohistochemistry, and RT-PCR and western blotting as visualization and quantification techniques respectively. In liver, lung, and kidney, TRalpha mRNA was expressed in the stromal and TRbeta mRNA in the parenchymal component of the tissues. When compared with liver, TRalpha mRNA concentrations were tenfold higher in lung, kidney, and intestine, and 100-fold higher in brain, with TRalpha (2) mRNA concentrations exceeding those of TRalpha (1) 5-to 10-fold. Tissue TRbeta (1) mRNA concentrations were similar in liver, lung, and brain, and 3- to 5-fold higher in kidney and intestine. None of the TRbeta (2) mRNA could be detected outside the pituitary. Tissue TRalpha (2) and TRbeta (1) protein levels reached adult levels at 5 days before birth, whereas TRalpha (1) protein peaked after birth. Because of the distinct time-course of thyroid hormone-binding receptors TRalpha (1) and TRbeta (1), we speculate that an initiating, TRbeta (1)-mediated signaling from the parenchyma is followed by a TRalpha (1)-mediated response in the stroma. When compared with organs with a complementary parenchymal-stromal expression pattern, organs with extensive cellular co-expression of TRalpha and TRbeta (brain and intestinal epithelium) were characterized by a very low TRalpha protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a complementary tissue expression pattern and in those with cellular co-expression of the TRalpha and TRbeta genes.
Electrospun matrices are proposed as an alternative for polypropylene meshes in reconstructive pelvic surgery. Here, we investigated the effect of fiber diameter on (1) the mechanical properties of ...electrospun poly (lactic-co-glycolic acid)-blended-poly(caprolactone) (PLGA/PCL) matrices; (2) cellular infiltration; and (3) the newly formed extracellular matrix (ECM) in vitro. We compared electrospun matrices with 1- and 8 μm fiber diameter and used nonporous PLGA/PCL films as controls. The 8-μm matrices were almost twice as stiff as the 1-μm matrices with 1.38 and 0.66 MPa, respectively. Matrices had the same ultimate tensile strength, but with 80% the 1-μm matrices were much more ductile than the 8-μm ones (18%). Cells infiltrated deeper into the matrices with larger pores, but cellular activity was comparable on both substrates. New ECM was deposited faster on the electrospun samples, but after 2 and 4 weeks the amount of collagen was comparable with that on nonporous films. The ECM deposited on the 1-μm matrices, and the nonporous film was about three times stiffer than the ECM found on the 8-μm matrices. Cell behavior in terms of myofibroblastic differentiation and remodeling was similar on the 1-μm matrices and nonporous films, in comparison to that on the 8-μm matrices. We conclude that electrospinning enhances the integration of host cells as compared with a nonporous film of the same material. The 1-μm matrices result in better mechanical behavior and qualitatively better matrix production than the 8-μm matrices, but with limited cellular infiltration. These data are useful for designing electrospun matrices for the pelvic floor.
This study focuses on the development of a universal mathematical model for drug release kinetics from liposomes to allow in silico prediction of optimal conditions for fine-tuned controlled drug ...release. As a prelude for combined siRNA-drug delivery, nanoliposome formulations were optimized using various mole percentages of a cationic lipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) in the presence or absence of 3 mol% distearoyl phosphoethanolamine, polyethylene glycol (PEG-2000mDSPE). Outcome parameters were particle size, zeta potential, entrapment efficiency, in vitro drug release, and tumor cell kill efficiency. The optimized formula (containing 20% DOTAP with 3% DSPE-mPEG(2000) was found to be stable for six months, with round-shaped particles without aggregate formation, an average diameter of 71 nm, a suitable positive charge, and 89% drug encapsulation efficiency (EE). The 41% drug release during 6 h confirmed controlled release. Furthermore, the release profiles as functions of pH and temperature were investigated and the kinetics of the drug release could adequately be fitted to Korsmeyer-Peppas' model by multiple regression analysis. The statistical parameters confirmed good conformity of final models. Functionality of the novel cationic liposome formulations (± DOX) was tested on osteosarcoma (OS) cell lines. Increased OS cell toxicity (1.3-fold) was observed by the DOX-loaded vs. the free DOX. A feasibility pilot showed that siRNA could be loaded efficiently as well. In conclusion, we have established a predictive mathematical model for the fine-tuning of controlled drug release from liposomal formulations, while creating functional drug-delivery liposomes with potential for siRNA co-delivery to increase specificity and efficacy.
Type II deiodinase (D2) plays a key role in regulating thyroid hormone-dependent processes in, among others, the central nervous system (CNS) by accelerating the intracellular conversion of T4 into ...active T3. Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid axis, D2 activity also appears to show daily variations. However, the mechanisms involved in generating these daily variations, especially in the CNS, are not known. Therefore, we decided to investigate the role the master biological clock, located in the hypothalamus, plays with respect to D2 activity in the rat CNS as well as the role of one of its main hormonal outputs, i.e. plasma corticosterone. D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Ablation of the biological clock abolished the daily variations of D2 activity in all four tissues studied. The main effect of the knockout of the suprachiasmatic nuclei (SCN) was a reduction of nocturnal peak levels in D2 activity. Moreover, contrary to previous observations in SCN-intact animals, in SCN-lesioned animals, the decreased levels of D2 activity are accompanied by decreased plasma levels of the thyroid hormones, suggesting that the SCN separately stimulates D2 activity as well as the hypothalamo-pituitary-thyroid axis.
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.
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The extracellular matrix of fascia-like tissues is a resilient network of collagenous fibers that withstand the forces of daily life. When overstretched, the matrix may tear, with ...serious consequences like pelvic organ prolapse (POP). Synthetic implants can provide mechanical support and evoke a host response that induces new matrix production, thus reinforcing the fascia. However, there is considerable risk of scar formation and tissue contraction which result in severe complications. Matrix producing fibroblasts are both mechanosensitive and contractile; their behavior depends on the implant’s surface texture and mechanical straining. Here we investigate the effect of both in a newly-designed experimental setting.
Electrospun scaffolds of Nylon and PLGA/PCL and a non-porous PLGA/PCL film were clamped like a drumhead and seeded with fibroblasts of POP patients. Upon confluency, scaffolds were cyclically strained for 24 or 72 h at 10% and 0.2 Hz, mimicking gentle breathing. Non-loading condition was control. Strained fibroblasts loosened their actin-fibers, thereby preventing myofibroblastic differentiation. Mechanical loading upregulated genes involved in matrix synthesis (collagen I, III, V and elastin), matrix remodeling (α-SMA, TGF-β1, MMP-2) and inflammation (COX-2, TNF-α, IL8, IL1-β). Collagen genes were expressed earlier under mechanical loading and the ratio of I/III collagen increased. Matrix synthesis and remodeling were stronger on the electrospun scaffolds, while inflammation was more prominent on the non-porous film. Our findings indicate that mechanical straining enhances the regenerative potential of fibroblasts for the regeneration of fascia-type tissues and limit the risk of scar tissue formation. These effects are stronger on an electrospun texture.
Pelvic organ prolapsed is a dysfunctional disease in female pelvic floor that can reduce the quality of life women. Currently, trans-vaginal knitted meshes are used to anatomically correct the dysfunctional tissues. However, the meshes can create sever adverse complications in some patients (e.g. chronic pain) in longer-term. As an alternative, we developed nanofibrous matrices by electrospinning based on different materials. We designed an in-vitro culture system and subjected cell-seeded matrices to cyclic mechanical loading. Results revealed that gentle straining of POP-cells on electrospun matrices, advances their regenerative potential at morphological and gene expression levels. Our findings, provide a proof-of-concept for using electrospun matrices as an alternative implant for pelvic floor repair, given that the parameters are designed efficiently and safely.
To assess the cytotoxicity of an experimental hybrid-glass-based infiltrant and its effect on biofilm attachment, growth and metabolic activity, and to compare it to the resin-based infiltrant Icon.
...Cytotoxicity of hybrid-glass-based material (EXP) and resin-based infiltrant Icon (Icon) was tested in direct contact tests on freshly cured (direct_mat) and on materials kept for 24 h in cell culture medium (direct_exmat), and extract test with materials 24-h extracts (extract). Cell viability of L929 mouse fibroblast cell line was measured with MTT assay, according to ISO10993–5:2009. Biofilm attachment (5 h), growth (24 h and 48 h) and lactic-acid production (24 h and 48 h) on glass-disk specimens coated with EXP or Icon, or uncoated (control), were assessed using a microcosm biofilm model and Amsterdam Active Attachment system. At indicated time points, biofilms were harvested, plated, and CFU counts were determined, while lactic-acid production was measured colorimetrically.
Cell viability reduction by EXP was below 30%-threshold in direct contact tests, while in extract test an increased cell viability was observed. Icon reduced cell viability substantially in all three tests. Significantly less bacteria attached to the surface of EXP after 5 h compared to Icon and control. Biofilm growth was significantly lower on EXP than on Icon and control after 24 h, but this difference was smaller and statistically insignificant after 48 h. There was no difference in lactic-acid production among groups.
Novel hybrid-glass-based infiltrant seems to have a better biocompatibility and accumulates on its surface less bacteria than resin-based infiltrant, which makes it an attractive resin-free alternative.
•Hybrid-glass-based infiltrant shows no cytotoxic effect on L929 fibroblasts.•Initial bacterial attachment and early biofilm growth are significantly lower on hybrid-glass-based infiltrant compared to resin-based infiltrant.•Hybrid-glass-based infiltrant is an attractive alternative to resin-based infiltrant.
Cationic liposomes have been investigated as non-viral vectors for gene delivery for more than a decade to overcome challenges associated with viral gene delivery. However, due to instability of ...liposomes, siRNA delivery is still a serious problem. In this study, we developed stealth PEGylated liposome formulations and focused on the effects of PEGylated liposomes on parameters related to size, zeta potential, polydispersity index, siRNA-loading efficiency and long-term stability of the siRNA-liposome complex. We were able to generate siRNA lipoplexes that could be very efficiently loaded, did not aggregate, could be stored at 4 °C for at least 6 months with only marginal release (1-5%) of siRNA and enhanced intracellular delivery of siRNA. Moreover, we could demonstrate that PEGylation positively contributed to all these parameters compared to liposomes, which were not PEGylated. The prepared lipoplex was successfully silenced J1P1 expression in MG-63 osteosarcoma cell line. In conclusion, our novel PEGylated liposomes have high potential for systemic delivery of siRNA and can improve in vivo stability of free siRNA and also siRNA lipoplexes.
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