Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive due to insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces.
Bone marrow macrophages (BMMO) and IC-21, RAW 264.7 and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pre-treated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking assessed integrins critical for monocyte/macrophage adhesion in vitro.
Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures pre-adsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared to Teflon® AF, plasma-polymerized FC was less permissive to extended cell proliferation. The β2 integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of pre-adsorbed protein biasing, with respect to adhesion, cell morphology, motility and proliferation.
perfluorocarbon; foreign body reaction; integrin blocking; cell line; serum proteins
Monocytes/macrophages and fibroblasts are recruited to the injury site and orchestrate the host response and tissue repair. We have previously shown that polyethylene glycol (PEG)-ylated arginine-glycine-aspartic acid (RGD) sequence grafted onto an extracellular matrix (ECM)-based semi-interpenetrating network (sIPN) enhances monocyte adhesion, and modulates subsequent gene expression and release of inflammatory and matrix remodeling factors. In this study, we investigate the direct influence of fibroblasts on monocyte response to this ECM mimic. Key wound-healing factors in inflammation, matrix remodeling, and regeneration were analyzed to gain insight into the interrelated role of regulation in fibroblast-monocyte interaction. Interleukin-1alpha/−1beta (IL-1α/−1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte inflammatory protein-1alpha/−1beta (MIP-1α/−1β), transforming growth factor-alpha (TGF-α), monocyte chemoattractant factor (MCP-1), matrix metalloproteinase-2/−9 (MMP-2/−9), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) were analyzed. Fibroblasts decreased monocyte adhesion onto the RGD-grafted sIPN while increasing monocyte GM-CSF on all surfaces over time except for on RGD and PHSRN-grafted sIPN at 96 h. Monocytes decreased initial fibroblast IL-1α and TGF-α, but drastically increased fibroblast MMP-2 and GM-CSF. Monocyte IL-1β, TNF-α, MIP-1β, MCP-1, MMP-9, and GM-CSF expression was increased over time in the presence of all sIPNs, and when the sIPNs were immobilized with ligands, a down-regulation of fibroblast IL-1β, MIP-1α, MIP-1β compared with unmodified sIPN was observed. When the ligand immobilized was RGD, monocyte TGF-α, MIP-1β, and VEGF expression was increased while monocyte GM-CSF was decreased at selected time points. These results showed a dynamic monocyte response to selected ECM components in the presence of fibroblasts.
monocytes/macrophages; fibroblast; interleukin-1; matrix metalloproteinase-2/-9; granulocyte-macrophage colony-stimulating factor; vascular endothelial growth factor
A polymerizable superoxide dismutase mimetic (SODm) was incorporated into poly(ethylene glycol) (PEG) hydrogels to protect encapsulated cells from superoxide-mediated damage. Superoxide and other small reactive oxygen species (ROS) can cause oxidative damage to donor tissue encapsulated within size exclusion barrier materials. To enzymatically breakdown ROS within biomaterial cell encapsulation systems, Mn(III) Tetrakis[1-(3-acryloxy-propyl)-4-pyridyl] porphyrin (MnTTPyP-acryl), a polymerizable manganese metalloporphyrin SOD mimetic, was photopolymerized with PEG diacrylate (PEGDA) to create functional gels. In unmodified PEG hydrogels, a significant reduction in metabolic activity was observed when encapsulated Min6 β-cells were challenged with chemically generated superoxide. Cells encapsulated within MnTPPyP-co-PEG hydrogels, however, demonstrated greatly improved metabolic activity following various superoxide challenges. Further, cells were encapsulated and cultured for 10 days within MnTPPyP-co-PEG hydrogels and challenged with superoxide on days 4, 6, and 8. At the conclusion of this study, cells in blank PEG hydrogels had no observable metabolic activity but when encapsulated in MnTPPyP-functionalized hydrogels, cells retained 60 ± 5% of the metabolic activity compared to untreated controls.
reactive oxygen species; superoxide; hydrogel; β-cell; superoxide dismutase
More than 400,000 primary hip and knee replacement surgeries are performed each year in the United States. From these procedures, approximately 0.5–3% will become infected and when considering revision surgeries, this rate has been found to increase significantly. Antibiotic resistant bacterial infections are a growing problem in patient care. This in vitro research investigated the antimicrobial potential of the polymer released, broad spectrum, Cationic Steroidal Antimicrobial-13 (CSA-13) for challenges against 5 × 108 colony forming units (CFU) of methicillin-resistant Staphylococcus aureus (MRSA). It was hypothesized that a weight-to-weight (w/w) concentration of 18% CSA-13 in silicone would exhibit potent bactericidal potential when used as a controlled release device coating. When incorporated into a polymeric device coating, the 18% (w/w) broad-spectrum polymer released CSA-13 antimicrobial eliminated 5 × 108 CFU of MRSA within 8 hours. In the future, these results will be utilized to develop a sheep model to assess CSA-13 for the prevention of perioperative device related infections in vivo.
Ceragenin; CSA-13; Antimicrobial agents; Drug delivery; Controlled release device
The ability to design biomaterials that interact with biological environments in a predictable manner necessitates an improved understanding of how surface chemistry influences events such as protein adsorption and cell adhesion. In this work, we examined mechanisms governing the interactions between 3T3 fibroblasts and nylon-3 polymers, which have a protein-like polyamide backbone and are highly amenable to tuning of chemical and physical properties. Protein adsorption and cell adhesion to a library of nylon-3 polymers were characterized and analyzed by partial least squares regression. This analysis revealed that specific chemical features of the nylon-3 polymers correlated with the extent of protein adsorption, which, in turn, correlated with cell adhesion in a serum-containing environment. In contrast, in a serum-free environment, cell adhesion could be predicted solely from chemical properties. Enzymatic treatments of 3T3 cells prior to plating indicated that proteins bound to the cell surface mediated cell-nylon-3 polymer interactions under serum-free conditions, with additional analysis suggesting that cell-associated fibronectin played a dominant role in adhesion in the absence of serum. The mechanistic insight gained from these studies can be used to inform the design of new polymer structures in addition to providing a basis for continued development of nylon-3 copolymers for tissue engineering applications.
Cell adhesion; Protein adsorption; Cell-material interactions; Nylon-3; PLSR
In vitro human mesenchymal stem cell (hMSC) proliferation and differentiation is dependent on scaffold design parameters and specific culture conditions. In this study, we investigate how scaffold microstructure influences hMSC behavior in a perfusion bioreactor system. Poly-l-lactic acid (PLLA) scaffolds are fabricated using supercritical carbon dioxide (SC-CO2) gel drying. This production method results in scaffolds fabricated with nanostructure. To introduce a microporous structure, porogen leaching was used in addition to this technique to produce scaffolds of average pore size of 100, 250, and 500 µm. These scaffolds were then cultured in static culture in well plates or dynamic culture in the tubular perfusion system (TPS) bioreactor. Results indicated that hMSCs were able to attach and maintain viability on all scaffolds with higher proliferation in the 250 µm and 500 µm pore sizes of bioreactor cultured scaffolds and 100 µm pore size of statically cultured scaffolds. Osteoblastic differentiation was enhanced in TPS culture as compared to static culture with the highest alkaline phosphatase expression observed in the 250 µm pore size group. Bone morphogenetic protein-2 was also analyzed and expression levels were highest in the 250 µm and 500 µm pore size bioreactor cultured samples. These results demonstrate cellular response to pore size as well as the ability of dynamic culture to enhance these effects.
supercritical fluids; scaffold; PLLA; human mesenchymal stem cells; tissue engineering; bioreactor
The clinical potential of siRNA based therapeutics remains hindered by the challenge of delivering enough siRNA into the cytoplasm to yield a clinically relevant effect. Although much research has focused on optimizing delivery vehicles for this class of molecules, considerably less is known about the microenvironmental influences on the response of target cells to siRNA. The substrate to which cells adhere is one component of the microenvironment that can modulate cellular behavior. Here, we tested the hypothesis that modulating the properties of cellular adhesion substrates can alter siRNA efficacy. Specifically, cationic lipid complexed siRNA particles were applied to U251 cells seeded on alginate hydrogel surfaces with systematic variation in elastic modulus and integrin ligand RGD density. These experiments revealed no change in siRNA mediated eGFP knockdown over the elastic modulus range tested (53 to 133 kPa). However, an eight-fold increase in RGD content of the alginate growth substrate resulted in an increase in siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, a more than two fold increase in silencing. Our results identify control of the cell-adhesion substrate interaction as a modulator of siRNA protein silencing efficacy.
siRNA; adhesion; hydrogel; modulus; RGD
The purpose of this study was to evaluate whether treatment time and concentration of these reagents have an effect on the resulting gliding resistance. Forty peroneus longus (PL) tendons were used, from 20 adult mongrel dogs, along with the A2 pulley obtained from the ipsilateral hind paw. After the baseline gliding resistance was measured, the PL tendons were treated with one of three concentrations of hyaluronic acid (HA) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS) mixed with 10% gelatin for various times (5, 30, and 60 min). Tendon friction was measured over 1000 cycles of simulated flexion/extension motion. Gliding resistance of the untreated PL tendons had no significant difference among the groups. After surface treatment with low concentration of HA and EDC/NHS for 5-min cure, the gliding resistance was similar to that of the untreated PL tendon and significantly higher than its 30- and 60-min treatment. For the rest of high concentration of HA and EDC/NHS groups, the gliding resistance was lower than that of untreated PL tendon. However, there was no significant difference among the timing points. It is possible to optimize the effect of surface treatment on friction and durability by regulating cure time and concentration of reagents in a canine extrasynovial tendon in vitro.
hyaluronic acid; flexor tendon; extrasynovial tendon; friction; chemical modification
Bone substitute materials are required to support the remodeling process, which consists of osteoclastic resorption and osteoblastic synthesis. Osteoclasts, the bone resorbing cells, generate from differentiation of hemopoietic mononuclear cells. In the present study we have evaluated the effects of 1.0 wt% strontium (Sr) and 1.0 wt% magnesium (Mg) doping in beta-tricalcium phosphate (β-TCP) on the differentiation of mononuclear cells into osteoclast-like cells and its resorptive activity. In vitro osteoclast-like cell formation, adhesion, and resorption were studied using osteoclast precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κβ ligand (RANKL). Osteoclast-like cell formation was noticed on pure and Sr doped β-TCP samples at day 8 which was absent on Mg doped β-TCP samples indicating decrease in initial osteoclast differentiation due to Mg doping. After 21 days of culture, osteoclast-like cell formation was evident on all samples with osteoclastic markers such as actin ring, multiple nuclei, and presence of vitronectin receptor αvβ3 integrin. After osteoclast differentiation, all substrates showed osteoclast-like cell mediated degradation, however; significantly restricted for Mg doped β-TCP samples. Our present results indicated substrate chemistry controlled osteoclast differentiation and resorptive activity which can be used in designing TCP based resorbable bone substitutes with controlled degradation properties.
Beta-tricalcium phosphate; Dopants; Osteoclast-like cells; Osteoclastogenesis; Resorption lacunae
This study reports on the use of a fibrinogen-derived peptide for the specific targeting and delivery of vancomycin to Staphylococcus epidermidis biofilms. One method by which S. epidermidis initially adheres to biomaterials uses the plasma protein fibrinogen as an intermediary, where the S. epidermidis surface protein SdrG binds to a short amino acid sequence near the amino terminus of the Bβ chain of fibrinogen. We mimicked this binding interaction and demonstrated the use of a synthetic fibrinogen-based β6-20 peptide to target and deliver vancomycin to S. epidermidis in vitro. The β6-20 peptide was synthesized and labeled with a nanogold probe, and its targeting capabilities were examined through the use of scanning electron microscopy. The Nanogold component was then replaced by vancomycin, utilizing a flexible, variable length poly(ethylene glycol) linker between the peptide and antibiotic to create the targeted vancomycin products, β6-20-PEGx-VAN. Initial binding to surface adherent S. epidermidis was increased in a concentration-dependent manner relative to vancomycin for all equivalent concentrations ≥4 μg/ml, with targeted vancomcyin content up to 22.9 times that of vancomycin alone. Retention of the targeted antibiotics was measured after an additional 24 hour incubation period, revealing levels 1.3 times that of vancomycin. The results demonstrate the improved targeting and retention of vancomycin within a biofilm due to the incorporation of a specific targeting motif.
Staphylococcus epidermidis; SdrG; β6-20 peptide; Targeted Delivery; Vancomycin
Graded bilayered glass-ceramic composite coatings on Ti6Al4V substrates were fabricated using an enameling technique. The layers consisted of a mixture of glasses in the CaO-MgO-Na2O-K2O-P2O5 system with different amounts of calcium phosphates (CPs). Optimum firing conditions have been determined for the fabrication of coatings having good adhesion to the metal, while avoiding deleterious reactions between the glass and the ceramic particles. The final coatings do not crack or delaminate. The use of high-silica layers (>60 wt % SiO2) in contact with the alloy promotes long-term stability of the coating; glass-metal adhesion is achieved through the formation of a nanostructured Ti5Si3 layer. A surface layer containing a mixture of a low-silica glass (~53 wt % SiO2) and synthetic hydroxyapatite particles promotes the precipitation of new apatite during tests in vitro. The in vitro behavior of the coatings in simulated body fluid depends both on the composition of the glass matrix and the CP particles, and is strongly affected by the coating design and the firing conditions.
glass coating; enameling; titanium alloy; calcium phosphate; bioactivity; hydroxyapatite
A model in vitro system was developed for eliciting classical (M1) activation of surface-adherent murine macrophages, which was then used to study the interaction of the M1 macrophages with Staphylococcus epidermidis. Glass substrata were first covalently grafted with a mixture of methoxy- and biotin-terminated silanated polyethylene glycol. Interferon (IFN)-γ and/or lipopolysaccharide (LPS), ligands known to induce the highly microbicidal M1 activation state in macrophages, were biotinylated and immobilized by way of a streptavidin intermediate to the biotin-PEG base substratum. Assessment of mouse bone marrow-derived macrophage (BMDM) interleukin (IL)-12(p40) and nitric oxide response to the fabricated surfaces confirmed that the model system achieved activation of adherent macrophage: IFN-γ-presenting surfaces primed cells for M1 activation, LPS-presenting surfaces elicited innate activation, and surface presenting a combination of IFN-γ and LPS induced M1 activation. The phagocytic and microbicidal capacity of activated, surface-adherent BMDM was evaluated using S. epidermidis, a bacterial species prevalent in implant-associated infections. Results indicate that M1 activation of implant-adherent macrophages trends towards diminishing their phagocytic capacity, but enhances their microbicidal capacity for S. epidermidis.
immunostimulation; infection; inflammation; macrophage; surface grafting
Endothelial cell (EC) adhesion, shear retention, morphology, and hemostatic gene expression on fibronectin (FN) and RGD fluorosurfactant polymer (FSP) coated expanded polytetrafluoroethylene (ePTFE) grafts were investigated using an in vitro perfusion system. ECs were sodded on both types of grafts and exposed to 8dyn/cm2 of shear stress. After 24h the EC retention on RGD FSP coated grafts was 59±14%, which is statistically higher than the 36±11% retention measured on FN grafts (p<0.02). Additionally, ECs on RGD-FSP exhibited a more spread morphology and oriented in the direction of shear stress, as demonstrated by actin fiber staining. This spread morphology has been previously observed in cells that are adapting to shear stress. Real time PCR for vascular cell adhesion molecule 1, tissue factor, tissue plasminogen activator, and inducible nitric oxide synthase indicated that the RGD FSP material did not activate the cells and that shear stress appears to induce a more vasoprotective phenotype, as shown by a significant decrease in VCAM-1 expression, compared with sodded grafts. RGD FSP coating allows for a cell layer that is more resistant to physiological shear stress, as shown by the increased cell retention over FN. This shear stable EC layer is necessary for in vivo endothelialization of the graft material, which shows promise to increase the patency of synthetic small diameter vascular grafts.
Endothelial Cells; ePTFE Vascular Grafts; Surface Modification; Shear Stress
The lack of integration between implants and articular cartilage is an unsolved problem that negatively impacts the development of treatments for focal cartilage defects. Many approaches attempt to increase the number of matrix-producing cells that can migrate to the interface, which may help to reinforce the boundary over time but does not address the problems associated with an initially unstable interface. The objective of this study was to develop a bio-adhesive implant to create an immediate bond with the extracellular matrix components of articular cartilage. We hypothesized that implant-bound CollageN Adhesion protein, CNA, would increase the interfacial strength between a poly(vinly alcohol), PVA, implant and articular cartilage immediately after implantation, without preventing cell migration into the implant. By way of a series of in vitro immunohistochemical and mechanical experiments, we demonstrated that: free CNA can bind to articular cartilage, implant-bound CNA can bind to collagen type II and that implants functionalized with CNA result in a four-fold increase in interfacial strength with cartilage relative to un-treated implants at day zero. Of note, the interfacial strength significantly decreased after 21 days in culture which may be an indication that the protein itself has lost its effectiveness. Our data suggests that functionalizing scaffolds with CNA may be a viable approach towards creating an initially stable interface between scaffolds and articular cartilage. Further efforts are required to ensure long-term interface stability.
Decades of contradictory results have obscured the exact role of adsorbed fibronectin in the adhesion of the bacterium, Staphylococcus epidermidis (S. epidermidis), to biomaterials. Here, the ability of adsorbed fibronectin (FN) or bovine serum albumin (BSA) to modulate S. epidermidis adhesion to various biomaterials is reported. FN or BSA were adsorbed in increasing surface densities up to saturated monolayer coverage onto various common biomaterials, including poly(ethylene terephthalate) (PET), fluorinated ethylene propylene (FEP), poly(ether urethane) (PEU), silicone, and borosilicate glass. Despite the wide range of surface characteristics represented, adsorption isotherms varied only subtly between materials for the two proteins considered. S. epidermidis adhesion to the various protein-coated biomaterials was quantified in a static-fluid batch adhesion assay. While slight differences in overall adherent cell numbers were observed between the various protein-coated substrata, all materials exhibited significant dose-dependent decreases in S. epidermidis adhesion with increasing adsorption of either protein (FN, BSA) to all surfaces. Results here indicate that S. epidermidis adhesion to FN-coated surfaces is not a specific adhesion (i.e., receptor:ligand) mediated process, as no significant difference in adhesion was found between FN- and BSA-coated materials. Rather, results indicate that increasing surface density of either FN or BSA actually inhibited S. epidermidis adhesion to all biomaterials examined.
Fibronectin; Bacterial adhesion; Infection; Adsorption; Biomaterials
In vivo the vasculature provides an effective delivery
system for cellular nutrients; however, artificial scaffolds have no such
mechanism, and the ensuing limitations in mass transfer result in limited
regeneration. In these investigations, the regional mass transfer properties
that occur through a model scaffold derived from the human umbilical vein (HUV)
were assessed. Our aim was to define the heterogeneous behavior associated with
these regional variations, and to establish if different decellularization
technologies can modulate transport conditions to improve microenvironmental
conditions that enhance cell integration. The effect of three decellularization
methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol
(ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic
activity were assessed. Results show that regional variation in tissue structure
and composition significantly affects both mass transfer and cell function.
ACE/EtOH decellularization was shown to increase albumin mass flux through the
intima and proximate-medial region (0–250 μm) when compared with
sections decellularized with TX100 or SDS; although, mass flux remained constant
over all regions of the full tissue thickness when using TX100. Scaffolds
decellularized with TX100 were shown to promote cell migration up to
146% further relative to SDS decellularized samples. These results show
that depending on scaffold derivation and expectations for cellular integration,
specificities of the decellularization chemistry affect the scaffold molecular
architecture resulting in variable effects on mass transfer and cellular
mass transfer; decellularization; ex vivo tissue scaffold; cell migration; diffusion
Cell–matrix interaction is a key regulator for controlling stem cell fate in regenerative tissue engineering. These interactions are induced and controlled by the nanoscale features of extracellular matrix and are mimicked on synthetic matrices to control cell structure and functions. Recent studies have shown that nanostructured matrices can modulate stem cell behavior and exert specific role in tissue regeneration. In this study, we have demonstrated that nanostructured phase morphology of synthetic matrix can control adhesion, proliferation, organization and migration of human mesenchymal stem cells (MSCs). Nanostructured biodegradable polyurethanes (PU) with segmental composition exhibit biphasic morphology at nanoscale dimensions and can control cellular features of MSCs. Biodegradable PU with polyester soft segment and hard segment composed of aliphatic diisocyanates and dipeptide chain extender were designed to examine the effect polyurethane phase morphology. By altering the polyurethane composition, morphological architecture of PU was modulated and its effect was examined on MSC. Results show that MSCs can sense the nanoscale morphology of biphasic polyurethane matrix to exhibit distinct cellular features and, thus, signifies the relevance of matrix phase morphology. The role of nanostructured phases of a synthetic matrix in controlling cell–matrix interaction provides important insights for regulation of cell behavior on synthetic matrix and, therefore, is an important tool for engineering tissue regeneration.
mesenchymal stem cell; polyurethane; cell–matrix interaction; cellular organization
Cellular encapsulation within alginate hydrogel capsules has broad applications in tissue engineering. In seeking to improve the inherent instability of ionically cross-linked alginate hydrogels, we previously demonstrated the covalent stabilization of Ba2+ cross-linked alginate-azide beads via chemoselective Staudinger ligation using a 1-methyl-2-diphenylphosphino-terephthalate (MDT) terminated poly(ethylene glycol) linker. In this study, we functionalized variant PEG, linear and branched, and alginate polymers with MDT groups to evaluate the effect of size, structural design, number of functional groups, and charge on the resulting hydrogel bead. All cross-linkers resulted in enhanced covalent stabilization of alginate beads, with significant decreases in swelling and resistance to dissolution via Ba2+ chelation. The MDT-functionalized alginate resulted in the most stable and homogeneous bead, with the most restrictive permeability even after EDTA exposure. Co-encapsulation of MIN6 cells within the cross-linked alginate hydrogel beads resulted in minimal effects on viability, whereas the degree of proliferation following culture varied with cross-linker type. Altogether, the results illustrate that manipulating the cross-linker structural design permits flexibility in resulting alginate beads characteristics. Covalent stabilization of alginate hydrogel beads with these chemoselective alginate and PEG-based cross-linkers provides a unique platform for cellular encapsulation.
Staudinger ligation; alginate; PEG; cross-linker design; encapsulation
Currently, the majority of animal models that are used to study biofilm-related infections utilize planktonic bacterial cells as initial inocula to produce positive signals of infection in biomaterials studies. However, the use of planktonic cells has potentially led to inconsistent results in infection outcomes. In this study, well-established biofilms of methicillin-resistant Staphylococcus aureus (MRSA) were grown and used as initial inocula in an animal model of a Type IIIB open fracture. The goal of the work was to establish, for the first time, a repeatable model of biofilm implant-related osteomyelitis wherein biofilms were used as initial inocula to test combination biomaterials. Results showed that 100% of animals that were treated with biofilms developed osteomyelitis, whereas 0% of animals not treated with biofilm developed infection. The development of this experimental model may lead to an important shift in biofilm and biomaterials research by showing that when biofilms are used as initial inocula, they may provide additional insights into how biofilm-related infections in the clinic develop and how they can be treated with combination biomaterials to eradicate and/or prevent biofilm formation.
Planktonic; Biofilm; Initial Inocula; Animal Model; Infection
Calcium phosphate cements have many desirable properties for bone tissue engineering, including osteoconductivity, resorbability, and amenability to rapid prototyping based methods for scaffold fabrication. In this study, we show that dicalcium phosphate dihydrate (DCPD) cements, which are highly resorbable but also inherently weak and brittle, can be reinforced with poly(propylene fumarate) (PPF) to produce strong composites with mechanical properties suitable for bone tissue engineering. Characterization of DCPD-PPF composites revealed significant improvements in mechanical properties for cements with a 1.0 powder to liquid ratio. Compared to non-reinforced controls, flexural strength improved from 1.80 ± 0.19 MPa to 16.14 ± 1.70 MPa, flexural modulus increased from 1073.01 ± 158.40 MPa to 1303.91 ± 110.41 MPa, maximum displacement during testing increased from 0.11 ± 0.04 mm to 0.51 ± 0.09 mm, and work of fracture improved from 2.74 ± 0.78 J/m2 to 249.21 ± 81.64 J/m2. To demonstrate the utility of our approach for scaffold fabrication, 3D macroporous scaffolds were prepared with rapid prototyping technology. Compressive testing revealed that PPF reinforcement increased scaffold strength from 0.31 ± 0.06 MPa to 7.48 ± 0.77 MPa. Finally, 3D PPF-DCPD scaffolds were implanted into calvarial defects in rabbits for 6 weeks. Although the addition of mesenchymal stem cells to the scaffolds did not significantly improve the extent of regeneration, numerous bone nodules with active osteoblasts were observed within the scaffold pores, especially in the peripheral regions. Overall, the results of this study suggest that PPF-DCPD composites may be promising scaffold materials for bone tissue engineering.
Plasma expanders such as dextran and hydroxyethyl starch (HES) are important components of solutions designed to maintain vascular volume in the clinical setting and to preserve organs ex vivo before transplantation. Here, we show that these polymers also exert stabilizing effects on engineered microvessels in microfluidic type I collagen and fibrin scaffolds. Standard growth media, which did not contain dextran or HES, led to severe leakage, vascular collapse, and catastrophic failure of perfusion. Remarkably, vessels that were provided with 3% dextran or 5% HES had few focal leaks, maintained adhesion to the scaffold, and were typically viable and patent for at least two weeks. We found that the junctional marker VE-cadherin localized to a wide band in the presence of plasma expanders, but only at concentrations that also stabilized vessels. In conjunction with a previous computational model (Wong et al., Biomaterials 31, 4706-4714 (2010)), our results suggest that plasma expanders stabilize microvessels via physical mechanisms that enhance VE-cadherin localization at junctions and thereby limit vascular leakiness.
Microvascular tissue engineering; dextran; hydroxyethyl starch; microfluidic hydrogel; perfusion
In a previous study, we reported the upper limit of Young’s modulus of the unprotected protein at the dentin/adhesive interface to be 2 GPa. In this study, to obtain a more exact value of the moduli of the components at the d/a interface, we used demineralized dentin collagen with and without adhesive infiltration. The prepared samples were analyzed using micro-Raman spectroscopy (µRS) and scanning acoustic microscopy (SAM). Using an Olympus UH3 SAM (Olympus Co., Tokyo), measurements were recorded with a 400 MHz burst mode lens (120° aperture angle; nominal lateral resolution, 2.5 µm). A series of calibration curves were prepared using the relationship between the ultrasonically measured elastic moduli of a set of known materials and their SAM response. Finally, both the bulk and bar wave elastic moduli were computed for a set of 13 materials, including polymers, ceramics, and metals. These provided the rationale for using extensional wave measurements of the elastic moduli as the basis for extrapolation of the 400 MHz SAM data to obtain Young’s moduli for the samples: E = 1.76 ± 0.00 GPa for the collagen alone; E = 1.84 ± 0.65 GPa for the collagen infiltrated with adhesive; E = 3.4 ± 1.00 GPa for the adhesive infiltrate.
scanning acoustic microscopy; Young’s modulus; demineralized dentin collagen; dentin adhesive; calibration curves
Electrospun poly(ester urethane)urea (PEUU) scaffolds contain complex multiscale hierarchical structures that work simultaneously to produce unique macrolevel mechanical behaviors. In this study, we focused on quantifying key multiscale scaffold structural features to elucidate the mechanisms by which these scaffolds function to emulate native tissue tensile behavior. Fiber alignment was modulated via increasing rotational velocity of the collecting mandrel, and the resultant specimens were imaged using SEM under controlled biaxial strain. From the SEM images, fiber splay, tortuosity, and diameter were quantified in the unstrained and deformed configurations. Results indicated that not only fiber alignment increased with mandrel velocity but also, paradoxically, tortuosity increased concurrently with mandrel velocity and was highly correlated with fiber orientation. At microlevel scales (1–10 μm), local scaffold deformation behavior was observed to be highly heterogeneous, while increasing the scale resulted in an increasingly homogenous strain field. From our comprehensive measurements, we determined that the transition scale from heterogenous to homogeneous-like behavior to be ~1 mm. Moreover, while electrospun PEUU scaffolds exhibit complex deformations at the microscale, the larger scale structural features of the fibrous network allow them to behave as long-fiber composites that deform in an affine-like manner. This study underscores the importance of understanding the structure–function relationships in elastomeric fibrous scaffolds, and in particular allowed us to link microscale deformations with mechanisms that allow them to successfully simulate soft tissue mechanical behavior.
tissue engineering; scaffolds; mechanics; elastomers; electrospun
Heart valve disease is a serious and growing public health problem for which prosthetic replacement is most commonly indicated. Current prosthetic devices are inadequate for younger adults and growing children. Tissue engineered living aortic valve conduits have potential for remodeling, regeneration, and growth, but fabricating natural anatomical complexity with cellular heterogeneity remain challenging. In the current study, we implement 3D bioprinting to fabricate living alginate/gelatin hydrogel valve conduits with anatomical architecture and direct incorporation of dual cell types in a regionally constrained manner. Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced modulus, ultimate strength, and peak strain reducing slightly over 7-day culture, while the tensile biomechanics of cell-laden hydrogels were maintained. Aortic valve conduits were successfully bioprinted with direct encapsulation of SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4±3.4% for SMC and 83.2±4.0% for VIC) within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth muscle actin when printed in stiff matrix, while VIC expressed elevated vimentin in soft matrix. These results demonstrate that anatomically complex, heterogeneously encapsulated aortic valve hydrogel conduits can be fabricated with 3D bioprinting.
tissue engineering; interstitial cell; smooth muscle; biomechanics; cell encapsulation
Noncemented implants are the primary choice for younger patients undergoing total hip replacements. However, the major concern in this group of patients regarding revision is the concern from wear particles, periimplant inflammation, and subsequently aseptic implant loosening. Macrophages have been shown to liberate gold ions through the process termed dissolucytosis. Furthermore, gold ions are known to act in an anti-inflammatory manner by inhibiting cellular NF-κB-DNA binding. The present study investigated whether partial coating of titanium implants could augment early osseointegration and increase mechanical fixation. Cylindrical porous coated Ti-6Al-4V implants partially coated with metallic gold were inserted in the proximal region of the humerus in ten canines and control implants without gold were inserted in contralateral humerus. Observation time was 4 weeks. Biomechanical push out tests and stereological histomorphometrical analyses showed no statistically significant differences in the two groups. The unchanged parameters are considered an improvement of the coating properties, as a previous complete gold-coated implant showed inferior mechanical fixation and reduced osseointegration compared to control titanium implants in a similar model. Since sufficient early mechanical fixation is achieved with this new coating, it is reasonable to investigate the implant further in long-term studies.
gold; implant; osseointegration; arthroplasty; experimental