published_or_final_version / abstract / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output. / text

Tissue engineering is a field devoted to the design and creation of replacement tissues with the ultimate goal of one day providing replacement organs. Traditional strategies to accomplish this through the bulk seeding of cells onto a single monolithic porous bio-scaffold are unable to realise a precise architecture, thus the inability to mimic the cells natural micro-environment found within the body. Bio-printing approaches are the current state of the art with the ability to accurately mimic the complex 3D hierarchical structure of tissue. However, a functional construct also requires high strength to provide adequate support in load bearing applications such as bone and cartilage tissue engineering, and to maintain the open geometry of a large intricate channel network, which is crucial for the transport of nutrients and wastes. Typical approaches utilise materials which have processing parameters more amendable for cell incorporation, thus they can be simultaneously deposited with scaffolding material. However, the resulting construct is typically of low strength. This thesis explores the automation of a printing and “tissue assembly” process with the ability to incorporate delicate cell aggregates or spheroids within a high strength bio-scaffold requiring harsh processing parameters, at precise locations. The 3D printed bio-scaffold has a lattice architecture which enables a frictional fit to be formed between the particle and scaffold, thus preventing egress. To achieve this the pore must be expanded before the delivery of a single 1mm particle. Novel subsystems were developed to automate this process and provide the ability to achieve scalable, flexible, complex constructs with accurate architecture. A system architecture employing the benefits of modularity was devised. The main subsystems developed were the singulation device, to ensure the separation of a single particle; the injection device, to deliver and seed particles into the scaffold, and the control system, to facilitate the operation of the devices. Three generations of singulation devices have been developed ranging from mechanical to fluid manipulation methods alone. The first prototype utilised mechanical methods, with simple control methods. However the inability to correctly position the lead particle within the singulation chamber, resulted in damage to the test alginate particles. In the second prototype a fully fluidics based device utilised two trapping sites to capture the leading particles. Singulation success rates of up to 88% was achieved. Higher rates were limited by the trapped particle’s interaction with the lagging particles during capture. In a similar concept to the second prototype, the third prototype utilised only a single trapped particle, and achieved much higher throughput, and 100% singulation accuracy. The injection device, utilised a conical expanding rod within a thin outer sheath. It was able to expand the pore, with minimal damage to the scaffold, providing an unobstructed path for the delivery of the particle into the pore. A decentralised control system was devised to integrate the process operation for the electro-mechanical devices. Separate microcontrollers were able to sense, interact and communicate with one another, and the master control PC, to execute specific tasks to automate the process. The development of systems to automate the process has addressed the ability to accurately incorporate delicate cells with a high strength bio-scaffold, and will enable the realisation and investigation of intricate complex constructs, unachievable with current manual processes. Thus features found within the body may be more closely mimicked and functionalised, which may provide the necessary signals, micro-environment and infrastructure to correctly regulate the formation of complex functional tissue, supported by the adequate mass transport of nutrients and wastes. This may one day lead to 3D printing or assembly of viable replacement tissue, accurate in vitro model systems for laboratory testing, or even whole organs.

The integration of functional and structural properties makes genetically engineered proteins appealing in tissue engineering. Silk-elastinlike proteins (SELPs), containing tandemly repeated polypeptide sequence derived from natural silk and elastin, are recently under active study due to the interesting structure. The biological, chemical, physical properties of SELPs have been extensively investigated for their possible applications in drug/gene delivery, surgical tissue sealing and spine repair surgery. However, the mechanical aspect has rarely been looked into. Moreover, many other biomaterials have been fabricated into fibers in micrometer and nanometer scale to build extracellular matrix-mimic scaffolds for tissue regeneration, but many have one or mixed defects such as: poor strength, mild toxicity or immune repulsion etc. The SELP fibers, with the intrinsic primary structures, have novel mechanical properties that can make them defects-minimized scaffolds in tissue engineering.In this study, one SELP (SELP-47K) was fabricated into microfibers and nanofibers by the techniques of wet-spinning and electrospinning. Microfibers of meters long were formed and collected from a methanol coagulation bath, and later were crosslinked by glutaraldehyde (GTA) vapor. The resultant microfibers displayed higher tensile strength up to 20 MPa and higher deformability as high as 700% when tested in hydrated state. Electrospinnig of SELP-47K in formic acid and water resulted in rod-like and ribbon-like nanofibrous scaffolds correspondingly. Both chemical (methanol and/or GTA) and physical (autoclaving) crosslinking methods were utilized to stabilize the scaffolds. The chemical crosslinked hydrated scaffolds exhibit elastic moduli of 3.4-13.2 MPa, ultimate tensile strength of 5.7-13.5 MPa, and deformability of 100-130%, closely matching or exceeding the native aortic elastin; while the autoclaved one had lower numbers: 1.0 MPa elastic modulus, 0.3 MPa ultimate strength and 29% deformation. However, the resilience was all above 80%, beyond the aortic elastin, which is 77%. Additionally, Fourier transform infrared spectra showed clear secondary structure transition after crosslinking, explaining the phenomenon of scaffold water-insolubility from structural perspective and showed a direct relationship with the mechanical performance. Furthermore, the in vitro biocompatibility of SELP-47K nanofibrous scaffolds were verified through the culture of NIH 3T3 mouse embryonic fibroblast cells.

In the field of biomaterials, naturally-derived and synthetic polymers are utilized individually or in combination with each other, to create bio-inspired or biomimetic materials for various bioengineering applications, including drug delivery and tissue engineering. Natural polymers, such as proteins and polysaccharides, are advantageous due to low or non-toxicity, sustainable resources, innocuous byproducts, and cell-instructive properties. Synthetic polymers offer a variety of controlled chemical and physical characteristics, with enhanced mechanical properties. Together, natural and synthetic polymers provide an almost endless supply of possibilities for the development of novel, smart materials to resolve limitations of current materials, such as limited resources, toxic components and/or harsh chemical reactions. Herein is discussed the synthetic-biological material formation for cell-instructive tissue engineering and controlled drug delivery. We hypothesized that the combination of hydrogel-based scaffold and engineered nanomaterials would assist in the development or regeneration of tissue and disease treatment. Chemically-modified alginate was formed into alginate-based nanoparticles (ABNs) to direct the intracellular delivery of proteins (e.g., growth factors) and small molecular drugs (e.g., chemotherapeutics). The ABN surface was modified with cell-targeting ligands to control drug delivery to specific cells. The ABN approach to controlled drug delivery provides a platform for studying and implementing non-traditional biological pathways for disease (e.g., osteoporosis, multiple sclerosis) and cancer treatment. Through traditional organic and polymer chemistry techniques, and materials engineering approaches, a stimuli-responsive alginate-based smart hydrogel (ASH) was developed. Physical crosslinks formed based on supramolecular networks consisting of β-cyclodextrin-alginate and a tri-block amphiphilic polymer, which also provided a reversible thermo-responsiveness to the hydrogel. The hydrogel was shear-thinning, and recovered physical crosslinks, i.e., self-healed, after un-loading. The ASH biomaterials provide a platform for injectable, therapeutics for tissue regeneration and disease treatment. Indeed, various hydrogel constituents and tunable mechanical properties created cell-instructive hydrogels which promoted tissue formation.

Mechanical stimulation, in combination with biochemical factors, is likely to be essential to the appropriate function of stem cells and the development of tissue engineered constructs for orthopaedic and other uses. A multi-axial bioreactor was designed and built by Bose ElectroForce to simulate physiologically relevant loading conditions of the intervertebral disc (IVD), including axial compression, hydrostatic pressure and perfusion flow to multiple constructs under the control of a software program. This research optimises the design and configuration of the perfusion system of the bioreactor and presents results of preliminary experimental work on the combined effects of axial compression and perfusion on the viability of mesenchymal stem cells encapsulated in alginate hydrogels and the ability of the cells to produce extracellular matrix (ECM). The results of this thesis illustrated the power of a design of experiments (DOE) approach as a troubleshooting quality tool. With a modest amount of effort, we have gained a better understanding of the perfusion process of the tri-axial bioreactor, improved operational procedures and reduced variation in the process. Furthermore, removing unnecessary tubing lengths, equipment and fittings has made cost savings. The steady flow energy equation (SFEE) was used to develop a numerical analysis framework that provides an insight into the balance between velocity, elevation and friction in the flow system. The pressure predictions agreed well with experimental data, thus validating the SFEE for fluid analysis in the bioreactor system. The numerical predictions can be used to estimate the pressures around the three-dimensional constructs with a given arrangement of the tubing and components of the bioreactor. The system can potentially support long-term cultures of cell-seeded constructs in controlled environmental conditions found in vivo to study the mechanobiology of nucleus pulposus tissue engineering and the aetiology of IVD degeneration. However, dynamic compression and perfusion with associated hydrostatic pressurization of culture medium resulted in significant loss of cell viability compared to the unstimulated controls. Due to a large number of factors affecting cell behaviour in the tri-axial bioreactor system, it is difficult to identify the exact parameters influencing the observed cell response. A strategy that could help to distinguish the effects of mechanical stimuli and specific physiochemical factors should combine experiments with mathematical modelling approaches, and use the sensing incorporated in the bioreactor design and process-control systems to monitor and control specific culture parameters. Optimisation of the cell passage and cell seeding density were identified as key areas to improve the production of GAG in future studies; since the production of ECM was not observed in both static and dynamic cultures. Further studies could also attempt to use other hydrogel scaffolds, such as agarose, which has been widely used in cartilage tissue engineering studies and hyaluronic acid - a component of the nucleus pulposus ECM.

This study focused on the comparison of the electrospun silk scaffolds to the electrospun silk fibroin gel scaffolds. Moreover, this study examined the differences in cross-linking effects of genipin and methanol as well as solvents on the mechanical properties and cell compatibility of the scaffolds. Silk scaffolds were electrospun from an aqueous solution or 1,1,1,3,3-hexafluoro-2-propanol (HFIP) without genipin, immediately after 8 % (wt) genipin was added to the solution, and 18 hours after genipin blended with the solution. Uniaxial tensile testing determined that the silk scaffolds electrospun from water exhibit a higher modulus and peak stress than that of the silk scaffolds electrospun from HFIP. In vitro cell culture was conducted to determine the cell compatibility of the various silk fibroin-based scaffolds. 4'-6-Diamidino-2-phenylindole (DAPI) staining and histology suggest that genipin may enhance cell compatibility, and that neither ethanol nor methanol inhibit cell interactions.

Tissue engineering is an interdisciplinary field that aims to restore, maintain, or improve diseased or damaged tissues. Electrospinning has become one of the most popular means to fabricate a scaffold for various tissue engineering applications as the process is extremely versatile and inexpensive. The ability for electrospinning to consistently create nanofibrous structures capable of mimicking the native extracellular matrix (ECM) is the basis behind why this technique is so successful in tissue engineering. Cardiovascular disease has been the leading cause of death in the United States for over 100 years, and because of this, the need for coronary artery replacements is in serious demand. More specifically, small diameter vessels (<6 mm I.D.) are most needed, due to the fact that they are most often affected and the current clinical replacements provide less than optimal long-term patency and regenerative ability. Tissue engineering of vascular grafts has been investigated for over 50 years, however, synthetic replacements made of Dacron® and expanded-poly(tetrafluoroethylene) (e-PTFE) still remain the clinical standard. This study examines a variety of different ways to alter different characteristics of electrospun constructs, to create scaffolds that would be favorable for use as a blood vessel replacement; the end goal being the creation of an acellular bioresorbable vascular graft that would provide sufficient mechanical support to withstand physiological forces, as well as ample biocompatibility to allow host cells to infiltrate and regenerate the graft as the structure degrades. As a way of tailoring the mechanical and thermal properties of a scaffold to be more conducive to that of a native artery, a novel co-polymer was created from the random copolymerization of two monomers; 1,4-Dioxan-2-one (DX) and DL-3-methyl-1,4-dioxan-2-one (DL-3-MeDX) were mixed at different ratios and electrospun, forming nanofibrous scaffolds that exhibited different mechanical and thermal properties. Next, scaffolds were electrospun from natural and synthetic polymers, and the potential for these materials to elicit the formation of an acute thrombotic occlusion was investigated by quantifying tissue factor expression from monocytes using a novel technique. Tissue factor expression by monocytes on the electrospun natural and synthetic polymer scaffolds was compared to that of e-PTFE to determine their potential for use as vascular graft materials. Platelet-rich plasma (PRP), a naturally occurring blood component which is comprised of supraphysiologic concentrations of autologous growth factors, was activated and lyophilized to form a preparation rich in growth factors (PRGF). PRGF was electrospun for the first time, to create a scaffold that would mimic the role of the native ECM in the wound healing cascade. Characterization of these scaffolds proved their bioactivity was enhanced, with cell infiltration occurring throughout the structures in as little as 3 days. Lastly, PRP/PRGF and/or heparin were incorporated into electrospun PCL scaffolds as a means of enhancing the regenerative potential and reducing the thrombogenic potential of the scaffolds, while supplying the constructs with mechanical stability. The release of several pro-regenerative growth factors and chemokines from the PRP incorporated scaffolds was analyzed and the effect of PRP and heparin on scaffold degradation characteristics was determined. Additionally, cell proliferation, migration, sprout formation, and chemokine release were evaluated, and results from these experiments proved the addition of PRP could enhance the regenerative potential of the electrospun scaffolds. The results from this study reveal the variety of ways in which a number of characteristics of an electrospun scaffold can be altered to create a more ideal bioresorbable vascular graft that has the potential to be regenerated within the body, while providing enough mechanical support for this to occur over time.

Tissue engineering scaffolds come in many shapes and sizes, however, due to difficulty manufacturing the microstructure architecture required in tissue engineering, most scaffolds are architecturally non-dynamic in nature. Because the microstructural architecture of all biological tissues is inherently complicated, non-dynamic tissue engineering scaffolds tend to be a poor platform for tissue regeneration. The current method for manufacturing dynamic tissue engineering scaffolds involves electrospinning successive layers of different fibers, an approach that exhibits no fiber transition between layers and subsequent delamination problems. In this study we aim to address the design challenges of tissue engineering scaffolds through our novel integrated fiber electrospinning technique. Developed in our lab, this electrospinning technique makes it possible to manufacture complex electrospun scaffolds tailorable to specific tissue engineering needs while minimizing delamination tendencies. Our goal is to enhance the capabilities of the tissue engineering field by increasing the manufacturable scaffold complexity and overall structural integrity of electrospun scaffolds.

Electrospinning is a technique used to generate scaffolds composed of nano- to micron-sized fibers for use in tissue engineering. This technology possesses several key weaknesses that prevent it from adoption into the clinical treatment regime. One major weakness is the lack of porosity exhibited in most electrospun scaffolds, preventing cellular infiltration and thus hosts tissue integration. Another weakness seen in the field is the inability to physically cut electrospun scaffolds in the frontal plane for subsequent microscopic analysis (current electrospun scaffold analysis is limited to sectioning in the cross-sectional plane). Given this it becomes extremely difficult to associate spatial scaffold dynamics with a specific cellular response. In an effort to address these issues the research presented here will discuss modifications to electrospinning technology, cryosectioning technology, and our understanding of cellular infiltration mechanisms into electrospun scaffolds. Of note, the hypothesis of a potentially significant passive phase of cellular infiltration will be discussed as well as modifications to cell culture protocols aimed at establishing multiple passive infiltration phases during prolonged culture to encourage deep cellular infiltration.