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Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation.
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Cisteína , Trombose , Animais , Anticorpos/metabolismo , Plaquetas/metabolismo , Cisteína/metabolismo , Maleimidas/metabolismo , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Trombose/metabolismo , Distribuição TecidualRESUMO
This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N-isopropylacrylamide)-based dual-network hydrogels loaded with poly( l -lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels-specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC-encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties.
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Cartilagem Articular/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrogênese , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual , Animais , Cartilagem Articular/citologia , Condrócitos/citologia , Técnicas de Cocultura , Células-Tronco Mesenquimais/citologia , CoelhosRESUMO
Extrusion bioprinted constructs for osteochondral tissue engineering were fabricated to study the effect of multi-material architecture on encapsulated human mesenchymal stem cells' tissue-specific matrix deposition and integration into an ex vivo porcine osteochondral explant model. Two extrusion fiber architecture groups with differing transition regions and degrees of bone- and cartilage-like bioink mixing were employed. The gradient fiber (G-Fib) architecture group showed an increase in chondral integration over time, 18.5 ± 0.7 kPa on Day 21 compared to 9.6 ± 1.6 kPa on Day 1 for the required peak push-out force, and the segmented fiber (S-Fib) architecture group did not, which corresponded to the increase in sulfated glycosaminoglycan deposition noted only in the G-Fib group and the staining for cellularity and tissue-specific matrix deposition at the fiber-defect boundary. Conversely, the S-Fib architecture was associated with significant mineralization over time, but the G-Fib architecture was not. Notably, both fiber groups also had similar chondral integration as a re-inserted osteochondral tissue control. While architecture did dictate differences in the cells' responses to their environment, architecture was not shown to distinguish a statistically significant difference in tissue integration via fiber push-out testing within a given time point or explant region. Use of this three-week osteochondral model demonstrates that these bioink formulations support the fabrication of cell-laden constructs that integrate into explanted tissue as capably as natural tissue and encapsulate osteochondral matrix-producing cells, and it also highlights the important role that spatial architecture plays in the engineering of multi-phasic tissue environments. STATEMENT OF SIGNIFICANCE: Here, an ex vivo model was used to interrogate fundamental questions about the effect of multi-material scaffold architectural choices on osteochondral tissue integration. Cell-encapsulating constructs resembling stratified osteochondral tissue were 3D printed with architecture consisting of either gradient transitions or segmented transitions between the bone-like and cartilage-like bioink regions. The printed constructs were assessed alongside re-inserted natural tissue plugs via mechanical tissue integration push-out testing, biochemical assays, and histology. Differences in osteochondral matrix deposition were observed based on architecture, and both printed groups demonstrated cartilage integration similar to the native tissue plug group. As 3D printing becomes commonplace within biomaterials and tissue engineering, this work illustrates critical 3D co-culture interactions and demonstrates the importance of considering architecture when interpreting the results of studies utilizing spatially complex, multi-material scaffolds.
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Bioimpressão , Células-Tronco Mesenquimais , Suínos , Humanos , Animais , Alicerces Teciduais , Engenharia Tecidual/métodos , Materiais Biocompatíveis/farmacologia , Cartilagem , Impressão Tridimensional , Bioimpressão/métodosRESUMO
Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.
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Cartilagem , Hidrogéis , Hidrogéis/farmacologia , Hidrogéis/química , Temperatura , Engenharia Tecidual , Polietilenoglicóis/química , Polímeros/química , Peptídeos/químicaRESUMO
Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.
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Osteotomia Mandibular , Procedimentos de Cirurgia Plástica , Humanos , Animais , Ovinos , Engenharia Tecidual , Retalhos Cirúrgicos/cirurgia , Mandíbula/cirurgia , Transplante ÓsseoRESUMO
Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study's aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.
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The field of biomaterials has advanced significantly in the past decade. With the growing need for high-throughput manufacturing and screening, the need for modular materials that enable streamlined fabrication and analysis of tissue engineering and drug delivery schema has emerged. Microparticles are a powerful platform that have demonstrated promise in enabling these technologies without the need to modify a bulk scaffold. This building block paradigm of using microparticles within larger scaffolds to control cell ratios, growth factors and drug release holds promise. Gelatin microparticles (GMPs) are a well-established platform for cell, drug and growth factor delivery. One of the challenges in using GMPs though is the limited ability to modify the gelatin post-fabrication. In the present work, we hypothesized that by thiolating gelatin before microparticle formation, a versatile platform would be created that preserves the cytocompatibility of gelatin, while enabling post-fabrication modification. The thiols were not found to significantly impact the physicochemical properties of the microparticles. Moreover, the thiolated GMPs were demonstrated to be a biocompatible and robust platform for mesenchymal stem cell attachment. Additionally, the thiolated particles were able to be covalently modified with a maleimide-bearing fluorescent dye and a peptide, demonstrating their promise as a modular platform for tissue engineering and drug delivery applications.
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The present study sought to demonstrate the swelling behavior of hydrogel-microcarrier composite constructs to inform their use in controlled release and tissue engineering applications. In this study, gelatin methacrylate (GelMA) and GelMA-gelatin microparticle (GMP) composite constructs were three-dimensionally printed, and their swelling and degradation behavior was evaluated over time and as a function of the degree of crosslinking of included GMPs. GelMA-only constructs and composite constructs loaded with GMPs crosslinked with 10 mM (GMP-10) or 40 mM (GMP-40) glutaraldehyde were swollen in phosphate-buffered saline for up to 28 days to evaluate changes in swelling and polymer loss. In addition, scaffold reswelling capacity was evaluated under five successive drying-rehydration cycles. All printed materials demonstrated shear thinning behavior, with microparticle additives significantly increasing viscosity relative to the GelMA-only solution. Swelling results demonstrated that for GelMA/GMP-10 and GelMA/GMP-40 scaffolds, fold and volumetric swelling were statistically higher and lower, respectively, than for GelMA-only scaffolds after 28 days, and the volumetric swelling of GelMA and GelMA/GMP-40 scaffolds decreased over time. After 5 drying-rehydration cycles, GelMA scaffolds demonstrated higher fold swelling than both GMP groups while also showing lower volumetric swelling than GMP groups. Although statistical differences were not observed in the swelling of GMP-10 and GMP-40 particles alone, the interaction of GelMA/GMP demonstrated a significant effect on the swelling behaviors of composite scaffolds. These results demonstrate an example hydrogel-microcarrier composite system's swelling behavior and can inform the future use of such a composite system for controlled delivery of bioactive molecules in vitro and in vivo in tissue engineering applications. Impact statement In this study, porous three-dimensional printed (3DP) hydrogel constructs with and without natural polymer microcarriers were fabricated to observe swelling and degradation behavior under continuous swelling and drying-rehydration cycle conditions. Inclusion of microcarriers with different crosslinking densities led to distinct swelling behaviors for each biomaterial ink tested. 3DP hydrogel and hydrogel-microcarrier composite scaffolds have been commonly used in tissue engineering for the delivery of biomolecules. This study demonstrates the swelling behavior of porous hydrogel and hydrogel-microcarrier scaffolds that may inform later use of such materials for controlled release applications in a variety of fields including materials development and tissue regeneration.
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Hidrogéis , Alicerces Teciduais , Gelatina , Impressão Tridimensional , Engenharia TecidualRESUMO
Osteochondral defects present a unique clinical challenge due to their combination of phenotypically distinct cartilage and bone, which require specific, stratified biochemical cues for tissue regeneration. Furthermore, the articular cartilage exhibits significantly worse regeneration than bone due to its largely acellular and avascular nature, prompting significant demand for regenerative therapies. To address these clinical challenges, we have developed a bilayered, modular hydrogel system that enables the click functionalization of cartilage- and bone-specific biochemical cues to each layer. In this system, the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) was click conjugated with either a cartilage- or bone-specific peptide sequence of interest, and then mixed with a suspension of thermoresponsive polymer and mesenchymal stem cells (MSCs) to generate tissue-specific, cell-encapsulated hydrogel layers targeting the cartilage or bone. We implanted bilayered hydrogels in rabbit femoral condyle defects and investigated the effects of tissue-specific peptide presentation and cell encapsulation on osteochondral tissue repair. After 12 weeks implantation, hydrogels with a chondrogenic peptide sequence produced higher histological measures of overall defect filling, cartilage surface regularity, glycosaminoglycan (GAG)/cell content of neocartilage and adjacent cartilage, and bone filling and bonding compared to non-chondrogenic hydrogels. Furthermore, MSC encapsulation promoted greater histological measures of overall defect filling, cartilage thickness, GAG/cell content of neocartilage, and bone filling. Our results establish the utility of this click functionalized hydrogel system for in vivo repair of the osteochondral unit. STATEMENT OF SIGNIFICANCE: Osteochondral repair requires mimicry of both cartilage- and bone-specific biochemical cues, which are highly distinct. While traditional constructs for osteochondral repair have mimicked gross compositional differences between the cartilage and bone in mineral content, mechanical properties, proteins, or cell types, few constructs have recapitulated the specific biochemical cues responsible for the differential development of cartilage and bone. In this study, click biofunctionalized, bilayered hydrogels produced stratified presentation of developmentally inspired peptide sequences for chondrogenesis and osteogenesis. This work represents, to the authors' knowledge, the first application of bioconjugation chemistry for the simultaneous repair of bone and cartilage tissue. The conjugation of tissue-specific peptide sequences successfully promoted development of both cartilage and bone tissues in vivo.
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Cartilagem Articular , Hidrogéis , Animais , Condrogênese , Peptídeos , Coelhos , Engenharia TecidualRESUMO
This work investigated the effect of poly(l-lysine) (PLL) molecular weight and concentration on chondrogenesis of cocultures of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) in PLL-loaded hydrogels. An injectable dual-network hydrogel composed of a poly(N-isopropylacrylamide)-based synthetic thermogelling macromer and a chondroitin sulfate-based biological network was leveraged as a model to deliver PLL and encapsulate the two cell populations. Incorporation of PLL into the hydrogel did not affect the hydrogel's swelling properties and degradation characteristics, nor the viability of encapsulated cells. Coculture groups demonstrated higher type II collagen expression compared to the MSC monoculture group. Expression of hypertrophic phenotype was also limited in the coculture groups. Histological analysis indicated that the ratio of MSCs to ACs was an accurate predictor of the degree of long-term chondrogenesis, while the presence of PLL was shown to have a more substantial short-term effect. Altogether, this study demonstrates that coculturing MSCs with ACs can greatly enhance the chondrogenicity of the overall cell population and offers a platform to further elucidate the short- and long-term effect of polycationic factors on the chondrogenesis of MSC and AC cocultures.
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Cartilagem Articular , Células-Tronco Mesenquimais , Diferenciação Celular , Células Cultivadas , Condrócitos , Condrogênese , Técnicas de Cocultura , Hidrogéis , PolilisinaRESUMO
Mandibular reconstruction requires functional and aesthetic repair and is further complicated by contamination from oral and skin flora. Antibiotic-releasing porous space maintainers have been developed for the local release of vancomycin and to promote soft tissue attachment. In this study, mandibular defects in six sheep were inoculated with 106 colony forming units of Staphylococcus aureus; three sheep were implanted with unloaded porous space maintainers and three sheep were implanted with vancomycin-loaded space maintainers within the defect site. During the same surgery, 3D-printed in vivo bioreactors containing autograft or xenograft were implanted adjacent to rib periosteum. After 9 weeks, animals were euthanized, and tissues were analyzed. Antibiotic-loaded space maintainers were able to prevent dehiscence of soft tissue overlying the space maintainer, reduce local inflammatory cells, eliminate the persistence of pathogens, and prevent the increase in mandibular size compared to unloaded space maintainers in this sheep model. Animals with an untreated mandibular infection formed bony tissues with greater density and maturity within the distal bioreactors. Additionally, tissues grown in autograft-filled bioreactors had higher compressive moduli and higher maximum screw pull-out forces than xenograft-filled bioreactors. In summary, we demonstrated that antibiotic-releasing space maintainers are an innovative approach to preserve a robust soft tissue pocket while clearing infection, and that local infections can increase local and remote bone growth.
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Mandíbula , Reconstrução Mandibular , Animais , Antibacterianos/uso terapêutico , Reatores Biológicos , Porosidade , Próteses e Implantes , OvinosRESUMO
Microfluidic devices have advanced significantly in recent years and are a promising technology for the field of tissue engineering. Highly sophisticated microfabrication techniques have paved the way for the development of complex ex vivo models capable of incorporating and measuring the real-time response of multiple cell types interacting together in a single system. Muscle-on-a-chip technology has drastically improved and serves as a drug screening platform for many muscular diseases such as muscular dystrophy, tendinosis, fibromyalgia, mitochondrial myopathy, and myasthenia gravis. This review seeks to communicate the gaps in knowledge of current muscular disease models and highlight the power of microfluidic devices in enabling researchers to better understand disease pathology and provide high throughput screening of therapeutics for muscular myopathies.
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Dispositivos Lab-On-A-Chip , Doenças Musculares/patologia , Engenharia Tecidual/instrumentação , Animais , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microtecnologia , Músculos/citologia , Músculos/patologia , Engenharia Tecidual/métodosRESUMO
Gene therapy holds great potential for conditions such as cardiovascular disease, including atherosclerosis and also vascular cancers, yet available vectors such as the adeno-associated virus (rAAV) transduce the vasculature poorly. To enable retargeting, a single-chain antibody (scFv) that binds to the vascular cell-adhesion molecule (VCAM-1) overexpressed at areas of endothelial inflammation was site specifically and covalently conjugated to the exterior of rAAV6. To achieve conjugation, the scFv was functionalized with an orthogonal click chemistry group. This conjugation utilized site-specific sortase A methodology, thus preserving scFv binding capacity to VCAM-1. The AAV6 was separately functionalized with 4-azidophenyl glyoxal (APGO) via covalent adducts to arginine residues in the capsid's heparin co-receptor binding region. APGO functionalization removed native tropism, greatly reducing rAAV6-GFP transduction into all cells tested, and the effect was similar to the inhibition seen in the presence of heparin. Utilizing the incorporated functionalizations, the scFv was then covalently conjugated to the exterior of rAAV6 via strain-promoted azide-alkyne cycloaddition (SPAAC). With both the removal of native heparin tropism and the addition of VCAM-1 targeting, rAAV6 transduction of endothelial cells was greatly enhanced compared to control cells. Thus, this novel and modular targeting system could have further application in re-directing AAV6 toward inflamed endothelium for therapeutic use.
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Nanomedicine holds great promise for vascular disease diagnosis and specific therapy, yet rapid sequestration by the mononuclear phagocytic system limits the efficacy of particle-based agents. The use of low-fouling polymers, such as poly(ethylene glycol), efficiently reduces this immune recognition, but these nondegradable polymers can accumulate in the human body and may cause adverse effects after prolonged use. Thus, new particle formulations combining stealth, low immunogenicity and biocompatible features are required to enable clinical use. Here, a low-fouling particle platform is described using exclusively protein material. A recombinant protein with superior hydrophilic characteristics provided by the amino acid repeat proline, alanine, and serine (PAS) is designed and cross-linked into particles with lysine (K) and polyglutamic acid (E) using mesoporous silica templating. The obtained PASKE particles have low-fouling behavior, have a prolonged circulation time compared to albumin-based particles, and are rapidly degraded in the cell's lysosomal compartment. When labeled with near-infrared fluorescent molecules and functionalized with an anti-glycoprotein IIb/IIIa single-chain antibody targeting activated platelets, the particles show potential as a noninvasive molecular imaging tool in a mouse model of carotid artery thrombosis. The PASKE particles constitute a promising biodegradable and versatile platform for molecular imaging of vascular diseases.
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Imagem Molecular , Proteínas/química , Trombose/diagnóstico por imagem , Animais , Incrustação Biológica , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Polietilenoglicóis/química , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Propriedades de SuperfícieRESUMO
Polymerized high internal phase emulsions (polyHIPEs) are highly porous constructs currently under investigation as tissue engineered scaffolds. We previously reported on the potential of redox-initiated polyHIPEs as injectable bone grafts that space fill irregular defects with improved integration and rapid cure. Upon subsequent investigation, the radical-initiated cure of these systems rendered them susceptible to oxygen inhibition with an associated increase in uncured macromer in the clinical setting. In the current study, polyHIPEs with increased resistance to oxygen inhibition were fabricated utilizing a tetrafunctional thiol, pentaerythritol tetrakis(3-mercaptoproprionate), and the biodegradable macromer, propylene fumarate dimethacrylate. Increased concentrations of the tetrathiol additive provided improved oxygen resistance as confirmed by polyHIPE gel fraction while retaining the requisite rapid cure rate, compressive properties, and pore architecture for use as an injectable bone graft. Additionally, thiol-methacrylate polyHIPEs exhibited increased degradation under accelerated conditions and supported critical markers of human mesenchymal stem cell activity. In summary, we have improved upon current methods of fabricating injectable polyHIPE grafts to meet translational design goals of improved polymerization kinetics under clinically relevant conditions without sacrificing key scaffold properties.