1,721,192 research outputs found
In vitro culturing human Mesenchymal Stem Cells on 3D scaffolds for bone tissue regeneration
No full textIn vitro culturing human Mesenchymal Stem Cells on 3D scaffolds for bone tissue regeneratio
Design and Characterization of a Thermogelling Ink and Support Bath System for Extrusion Bioprinting
No full textThree-dimensional (3D) printing methodologies are growing increasingly popular
for fabricating tissue engineering constructs, due to their ability to efficiently and
repeatably produce highly complex and patient-specific designs. Of particularly common
use is extrusion printing, as it can be used with many pre-existing hydrogel materials due
to the more readily compatible rheological and chemical requirements. However, extrusion
printing is also associated with the lowest resolution and cellular compatibility of the 3D
printing methodologies. Recent innovations in hydrogel support baths have addressed
some of these concerns, but the versatility of the studied support materials is limited, as is
the understanding of their physical relationship with the ink they support. This work
presents the adaptation of a poly(N-isopropylacrylamide)-based thermogelling macromer
(TGM) for extrusion printing through the inclusion of a poloxamer hydrogel support bath.
Material and instrument factors were assessed for their effect on printability and fiber size.
Due to the unique dual-gelling nature of the TGM ink, uniform scaffolds can be fabricated
consisting of TGM hydrogel fibers with tunable diameters between 80 and 200 μm. In
addition, material and instrument factors were also studied for their effect on printed
chondrocyte viability. It was determined that printed TGM scaffolds could support viable
chondrocytes while maintaining good resolution and uniformity. Finally, the physical
relationship between the ink and the support bath, and the dynamic nature of the bath’s
rheological properties, were illuminated, contributing to our understanding of this
bioprinting methodology
Biodegradable Hydrogel Composites for Growth Factor and Stem Cell Delivery in Osteochondral Tissue Engineering
No full textCartilage has a limited endogenous ability for self-repair and current clinical treatments for damaged or diseased cartilage tissue are insufficient. Additionally, there is a biological and mechanical interplay between cartilage and the underlying subchondral bone, linking the pathogenesis/regeneration of both tissues. Thus, this thesis seeks to develop hydrogel composites as growth factor and cell delivery vehicles to study the regeneration of osteochondral tissue. First, we investigated the release of growth factors from acellular hydrogel composites containing gelatin microparticles (GMPs) to stimulate the repair of cartilage tissue in an in vivo osteochondral defect model. Transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) were delivered from the chondral layer of bilayered hydrogel composites while the subchondral layer remained growth factor-free. Results demonstrated that dual delivery of TGF-β3 and IGF-1 did not synergistically enhance cartilage repair, regardless of release kinetics, and the delivery of IGF-1 alone positively stimulated osteochondral tissue repair. Subsequently, we focused on improving the repair of the subchondral bone. The second part of this thesis investigated the delivery of IGF-1 and bone morphogenetic protein-2 (BMP-2) from the chondral and subchondral layers, respectively, of bilayered scaffolds in vivo. Results showed that BMP-2 enhanced subchondral bone repair, and that while the dual delivery of both growth factors did not improve cartilage repair, they synergistically enhanced subchondral bone formation over the delivery of IGF-1 alone. Using the results from this study, we also investigated relationships between specific cartilage and bone repair metrics to provide a fuller understanding of the osteochondral repair process. Correlation analysis revealed an intrinsic association between the degree of subchondral bone formation and cartilage surface regularity. Lastly, the third part of this thesis investigated the hydrogel composites as stem cell delivery vehicles. Degradable GMPs were used as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs and subsequently encapsulated in hydrogels to investigate their role on influencing MSC differentiation and aggregation. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Results revealed that MSC-seeded GMPs exhibited more cell-cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Overall, these hydrogel composites demonstrate potential as growth factor and cell delivery vehicles for the stimulation and study of osteochondral tissue regeneration
Development of injec table nanocomposite scaffolds of single -walled carbon nanotubes and biodegradable polymers for bone tissue engineering
No full textNanocomposites based on single-walled carbon nanotubes (SWNTs) and poly(propylene fumarate) (PPF) were developed and characterized as injectable scaffold materials for bone tissue engineering. Similar to other synthetic biodegradable polymers, PPF lacks the mechanical properties required for regeneration of load-bearing bone tissue. SWNTs were applied as reinforcing agents because of their extremely high mechanical properties and aspect ratio. An effective load transfer from polymer matrix to SWNTs is needed for the mechanical reinforcement, which is challenged by the strong inter-tube aggregation of large SWNT bundles. Various methods including mechanical agitation, sonication, use of surfactants, and chemical functionalizations have been utilized to homogeneously disperse SWNTs in PPF. Characterized by melt-state rheology, mechanical testing, and electron microscopy, functionalized SWNTs (F-SWNTs) demonstrated excellent dispersion in PPF and a 2- to 3-fold increase in compressive and flexural mechanical properties with only 0.1 wt% loading concentration when compared to pure PPF. Another form of SWNTs, ultra-short SWNTs (US-tubes) with 20-80 nm length, demonstrated up to a 2-fold increase in mechanical properties over pure PPF and resulted in a less viscous nanocomposite for easier injection than uncut SWNTs. The in vitro cytocompatibility of these nanocomposites was evaluated based on cell response to their unreacted components, crosslinked networks, and degradation products. The results did not reveal any cytotoxicity for purified SWNTs, F-SWNTs, and US-tubes at 1-100 μg/mL concentrations. All three tested nanocomposites displayed nearly 100% cell viability and excellent cell attachment, indicating favorable cytocompatibility. Finally, scaffolds with porosity of 75-90 vol% were fabricated from nanocomposites of US-tubes and functionalized US-tubes using a thermal-crosslinking particulate-leaching technique. These highly porous scaffolds possessed nearly 100% interconnected pore structures. Mechanical properties of nanocomposite scaffolds were higher than or similar to those of PPF scaffolds for all the porosities examined. In vitro osteoconductivity of these nanocomposite scaffolds was supported by the excellent attachment and proliferation of bone marrow stromal cells. These results indicate the great potential of injectable SWNT/PPF nanocomposites as the basis for bone tissue engineering scaffolds
Targeted delivery of osteogenic drugs for bone tissue engineering
No full textTo create a more efficient and effective method of osteogenic drug delivery in vivo, drugs were modified with high calcium affinity moieties including pamidronate, poly(aspartic acid), and poly(glutamic acid). To test the initial hypothesis that modified drugs can demonstrate the same bone binding capabilities of pamidronate, poly(aspartic acid), and poly(glutamic acid), these motifs were conjugated to model peptides and exhibited high affinity to hydroxyapatite (HA).
An in vitro controlled release experiment was conducted for native and modified TP508. Native and modified TP508 drugs were loaded in PLGA-PEG microparticles. Porous PPF scaffolds were injected with these drug-loaded particles, and in some instances with HA microparticles (20-50 or 50-100 mum). Less mineral surface resulted in less binding of drugs after release from the PLGA-PEG carriers and therefore a greater release than with the large HA particles. A final study was performed in the presence of 383 ng/mL collagenase, which cleaved the TP508 from the bone-binding domains at the point of the degradable peptide linker sequence.
The dose effect of TP508 was established by delivering 0, 25, 50, and 100 mug TP508 loaded into PPF scaffolds and implanted in a sized rat cranial defect. After 4 weeks, microCT analysis of the skulls revealed a statistically significant increase in bone formation for the 50 mug dose compared to controls and the 25 mug dose. Based on these findings, an equivalent of 50 mug TP508 or modified drugs were delivered from PLGA-PEG microspheres in the presence of 20-50 mum HA microparticles in the PPF scaffolds' pore network, which revealed no significant differences between drug groups. These results were promising in that this strategy of drug modification had no apparent negative effect on the bioactivity of TP508. Another finding of this work was that the incorporation of HA into PPF composites resulted in significantly greater bone formation, even after subtraction of the initial amount of HA. The addition of this osteoconductive material stimulated an increase in new bone over 4 weeks for both the control and 50 mug TP508 groups
Development of Extracellular Matrix-Based Colloidal Inks for Cartilage Tissue Engineering
No full textAdditive manufacturing enables spatial control over bioactive molecules and cells to mimic native tissue architecture, but a lack of bioinks that balance biological relevance and printability limits its potential. Decellularized extracellular matrix retains native biochemical cues but suffers from poor mechanical stability, restricting its use in 3D printing. This work presents the development of composite colloidal inks using methacryloylated decellularized cartilage extracellular matrix nanoparticles blending with gelatin nanoparticles to improve both printability and biofunctionality. The resulting inks are shear-thinning, self healing, and UVcrosslinkable, enabling the fabrication of tunable 3D-printing scaffolds. These scaffolds supported human bone marrow mesenchymal stem cell chondrogenesis, evidenced by enhanced collagen deposition, upregulation of chondrogenic gene expression, and suppression of osteogenic markers expression without exogenous differentiation factors. This study also explored the use of machine learning approaches to predict the print quality of 3D printed poly(propylene fumarate) and to identify relationships between printing parameters and the print quality. Print speed and material composition had the greatest effect on scaffold quality. Additionally, this work examined printing consistency with colloidal inks. Unlike poly(propylene fumarate), the colloidal inks required real-time parameter adjustments to maintain print fidelity, likely due to pressure-induced phase separation. Overall, this research introduces a novel, biologically active, and customizable colloidal ink platform for cartilage tissue engineering and broadens understanding of print behavior in colloidal systems
Development of thermally-crosslinked hydrogels as injectable cell carriers for orthopaedic tissue engineering
No full textSynthetic hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) were developed and characterized as injectable cell carriers for orthopaedic tissue engineering. Through alteration of the poly(ethylene glycol) molecular weight used in the synthesis of the OPF macromer, swelling and mechanical properties of the resulting crosslinked hydrogels could be controlled. These hydrogels were characterized, in both the swollen and dry states, leading to calculation of their mesh sizes, which varied depending on the OPF type used in crosslinking. In addition, it was found that these gels could be laminated during crosslinking, with each layer having distinct mechanical properties.
Before their use as injectable cell carriers, the cytotoxicity of all OPF hydrogel precursor molecules, including radical initiators and their derivatives, was evaluated using rat marrow stromal cells as a model cell type. Results indicated that the overall pH of the formulation, as well as length of exposure to the components, had significant effects on cell viability. Using this information, an initiator was identified which remained near neutral pH in cell culture media and resulted in crosslinking of two types of OPF hydrogels in 8 min at 37°C.
The optimized OPF formulations were then used to investigate effects of changes in hydrogel swelling properties and media supplements on osteogenic differentiation of encapsulated rat marrow stromal cells. After 28 days of in vitro culture, evidence of cellular differentiation was found in all sample types, indicating that the encapsulation procedure did not have a detrimental effect on the ability of the marrow stromal cells to form bone-like tissue. In the presence of osteogenic supplements, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less. In all specimens examined, areas of mineralized matrix were obvious many microns away from the cells, indicating that the hydrogel mesh size was large enough to allow diffusion of matrix components throughout the material. These results demonstrate the great potential of OPF hydrogels as injectable carriers for delivery of cells to a variety of complex orthopaedic defects
Fabrication of Three-Dimensional Printed Gradient Scaffolds for Heterogeneous Tissue Repair
No full textBone and cartilage damage are among the most common causes of disability among adults in the United States. Osteochondral defects, with limited self-repair capacity, remain a challenge as the cartilage-bone interface is highly complex. Native bone is likewise heterogeneous with localized structure and composition differences. A variety of clinical techniques have been used to treat these defects; however, these methods have several limitations, including donor site morbidity and the formation of immature tissue. Advances in tissue engineering have produced several strategies to address these challenges. However, many have similarly been limited by an inability to produce mechanically sufficient tissue or to mimic native heterogeneity. Others are cost prohibitive or technically challenging. The emergence of three-dimensional printing (3DP) has led to a variety of approaches that address highly heterogeneous tissues, allowing researchers to generate multiphasic constructs that recreate complex architectural and compositional gradients.
Here, we leveraged extrusion 3DP to develop multi-material constructs for tissue engineering. In Specific Aim 1, we fabricated multilayered polycaprolactone (PCL)-based constructs incorporating hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) to develop structures with porosity and composition gradients and evaluated resulting mechanical properties. In Specific Aim 2, gelatin methacrylate (GelMA)-based scaffolds containing gelatin microparticles (GMPs) were printed to demonstrate bulk hydrogel and hydrogel-microcarrier swelling. In Specific Aim 3, induction of cellular response was evaluated. PCL-HA scaffolds were fabricated with gradients in architecture and β-TCP content, and in vitro osteogenic differentiation of seeded MSCs was evaluated. Finally, PCL-HA was co-printed with GelMA-β-TCP to evaluate in vitro differentiation of seeded MSCs due to the ceramic concentration in the hydrogel phase.
In summary, this thesis details the development of 3D printed synthetic polymer-hydrogel-ceramic composite constructs for use in bone and osteochondral tissue engineering. We demonstrate the ability to create scaffolds with architectural and compositional gradients and their resulting mechanical properties. Additionally, we demonstrate the potential for these scaffolds to induce osteogenic differentiation. The results of this thesis lay the groundwork for future development of composite 3DP constructs in bone and osteochondral tissue engineering
Harnessing Inflammatory Signaling to Promote Bone Regeneration and Mitigate Joint Damage
No full textOnly volume 2 has been digitized.Inflammatory processes are infamous for their destructive effects on tissues and joints in a variety of diseases. Within the body, inflammation is a highly regulated biological response whose purpose is to promote tissue regeneration following injury. However, in certain settings, inflammation persists and leads to progressive tissue destruction. This thesis focused on modulating inflammatory signaling in both contexts. Part I investigated the effects of a model pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), on the in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). In contrast, Part II describes the development and in vivo evaluation of the first intra-articular controlled release system for the temporomandibular joint (TMJ), which silences inflammatory signaling and thus mitigates the painful joint damage seen in inflammatory TMJ disease. The following specific aims were addressed: (1) to determine the concentration of TNF-α that enhances in vitro osteogenic differentiation of MSCs; (2) to determine the temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (3) to determine the impact of bone-like extracellular matrix (ECM) on the concentration and temporal pattern of TNF-α delivery that enhances in vitro osteogenic differentiation of MSCs; (4) to evaluate the biocompatibility of intra-articular microparticles in the rat TMJ; (5) to develop a microparticle-based formulation for sustained release of a model anti-inflammatory small interfering ribonucleic acid (siRNA); and (6) to evaluate the therapeutic efficacy of intra-articular microparticles delivering siRNA in an animal model of TMJ inflammation. These studies led to the development of powerful strategies to rationally control inflammation to promote bone regeneration and mitigate joint damage in the setting of disease, both of which will ultimately improve the quality and specificity of therapies available in modern medicine
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