Matching Items (21)

In Vitro Osteogenic Study of hMSCs Under Diabetic Conditions

Description

Patients with type 2 diabetes mellitus experience a slower healing process and poor osteointegration, making it difficult for them to heal properly after a bone fracture. This study aims to

Patients with type 2 diabetes mellitus experience a slower healing process and poor osteointegration, making it difficult for them to heal properly after a bone fracture. This study aims to compare the proliferation and differentiation of human mesenchymal stromal cells at different glucose concentrations, as well as with an advanced glycated end-product (AGE) concentration, to mimic a healthy, prediabetic, and diabetic environment in an in vitro model over several experiments. Each experiment was composed of treatment groups in either growth or osteogenic media, with varying levels of glucose concentration or an advanced glycated end-product concentration. The treatment groups were cultured in 24 well plates over 28 days with staining of FITC-maleimide, DAPI, or alkaline phosphatase conducted at varying time points. The plates were imaged, then analyzed in ImageJ and GraphPad Prism. The study supports that at 28 days in culture, the more glucose added to osteogenic media treatment groups, the lower the nuclear count. At 14 days the same is true of growth media treatment groups, though the trend does not persist until 28 days. It does not seem that cell surface area of osteogenic groups, and growth media treatment groups was affected by glucose level. At 14 days, the alkaline phosphatase expression was unaffected by glucose level. However, at the 28 day time point the higher the glucose level of osteogenic treatment groups, the less expression of alkaline phosphatase. The effect of the added AGE concentration on hMSC osteogenesis was inconclusive. Overall, this study enhanced understanding of the role that glucose and AGEs play in the bone healing process for diabetic patients, allowing for future improvements of biomaterials and engineered tissue.

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  • 2019-05

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Electrospinning Stimuli-Responsive Fibers at the Nanoscale as Functional Drug Delivery Mats

Description

The objective of this research is to create biodegradable mats with tunable characteristics such as fiber diameter and surface area. The drug delivery mats enable spatially controlled delivery of

The objective of this research is to create biodegradable mats with tunable characteristics such as fiber diameter and surface area. The drug delivery mats enable spatially controlled delivery of disease-specific therapeutics. Using a large electric potential to draw fibers from a solution flowing at a specific rate, the polymer fibers reach a grounded target several inches away. The biodegradable polymer used in this study was poly(lactic acid-co-glycolic acid) (PLGA). PLGA solutions ranging from 0.5 to 27 wt.% were prepared by dissolving the block copolymer in a solvent mixture containing tetrahydrofuran (THF) and dimethylformamide (DMF) at a 3:1 weight ratio. They were then electrospun at needle-to-target distances of 7, 14, and 18 cm and rates ranging from 0.8 to 4 mL/h. The range of voltage used was between 8 – 15 kV, which was based on the observation of the formation of a Taylor cone, largely affected by on the environment and weather (e.g., temperature and humidity in the lab). A 27 wt.% PLGA solution, electrospun at 1 mL/h at a voltage of 11.25 kV and needle-to-target distance of 14 cm produced uniform fibers with an average fiber diameter of 0.985 m. All other parameters outside the range given created beaded fibers. In addition, solution rheology was performed on some of the PLGA solution to measure viscosity, which is directly correlated to the fiber diameter of the electrospun mats. Observing the impact of solvent on fiber spinning and fiber diameter brings about many positive results in developing fully characterized and well-understood fibrous mats for drug delivery. The nanoscale fibers will be used as drug delivery mats and, therefore, the biodegradation kinetics of the polymers will be studied. Next, parameters of the polymers as well as the polymeric mats will be correlated to the degradation-mediated release of small molecule therapeutics (e.g., peptides, drugs, etc.) such that time-resolved dosing profiles can be created.

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  • 2016-12

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Modulating the Heat Shock Response in E. coli to Optimize Membrane Protein Expression

Description

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding membrane proteins lags since their over-expression often leads to cell toxicity and death.[3,4] It was hypothesized that membrane protein expression could be regulated and optimized by modifying the heat shock response of Escherichia coli (E. coli). To test this hypothesis, the membrane protein expression pathway was reprogrammed using gene-blocks that were antisense to vital membrane protein DNA and RNA binding-site sequences and included an IbpA-σ32 heat shock promoter. Anti-PBAD and anti-HtdR gene-blocks were designed to have antisense sequences to the DNA of the arabinose PBAD promotor and Haloterrigena turkmenica deltarhodopsin (HtdR) transmembrane protein respectively. These sequences were then employed to be cloned into a pMM102 vector and grown in NEB-5α E. coli cells.

Stable glycerol stocks of the pIbpA-antiPBAD and pIbpA-antiHtdR in BW25113 cells with either a pBLN200 or pHtdR200 plasmid were created. Then after inducing the cells with L-arabinose and 10mM all-trans retinal to allow for membrane protein expression, spectrophotometry was used to test the optical density of the cells at an absorbance of 600nm. Although general trends showed that the pHtdR200-pMM102 and pHtdR200-pIbpA cells had lower optical densities than the pBLN200 cells of all types, the results were determined to be statistically insignificant. Continuing, the pHtdR200 cells of all types showed a purple phenotype when spun down, as expected, while the cells with the pBLN200 plasmid had a colorless phenotype in pellet form. Further work will include cloning a GFP gene-block to test the ability of the anti-PBAD sequence in tuning the transcription of the GFP protein.

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Date Created
  • 2018-05

Reflections on Engineering School from Practicing Engineers

Description

This study was conducted to look for ways to improve engineering school in order to maximize student benefit. The results of the survey showed that additional communication and professional

This study was conducted to look for ways to improve engineering school in order to maximize student benefit. The results of the survey showed that additional communication and professional interaction lessons as well as more emphasis on software and programming languages would help prepare engineers for their careers. There was unanimous support of communication materials from survey respondents, with constructive confrontation and career path discussion receiving the most positive feedback. Due to the unanimous support of communications material, and the fact that short communications lessons could drive home key points without adding too much work to engineering students’ already busy schedules, two short lesson outlines for constructive confrontation and career path discussion were produced for this study.

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  • 2020-12

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Controlling Calcium Binding on NorHA Scaffolds using a Biomineralization Peptide

Description

The tendon-bone junction is essential for allowing humans to transfer mechanical loads during activities. When injury does occur to this important area, current surgical techniques improperly bypass important physical and

The tendon-bone junction is essential for allowing humans to transfer mechanical loads during activities. When injury does occur to this important area, current surgical techniques improperly bypass important physical and chemical gradients and do not restore proper function. It is essential to create tissue engineered scaffolds that create proper models for the region and induce healing responses for repair. To advance research into these scaffolds, electrospinning fibers and hydrogels made of norbornene functionalized hyaluronic acid (NorHA) were used to promote bone growth by adhering calcium to the material. To further improve calcium adherence, which is indicative of bone regions, a mineralization peptide was allowed to soak through the fibers. NorHA proved to be a suitable material for biomineralization experiments, showing slow calcium adherence within the first hour before accelerating in adherence over 24 hours in both fibers and hydrogels. When the mineralization peptide was implemented calcium adherence on fibers increased nearly eight times within the first 15 minutes of experimentation.

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Date Created
  • 2020-05

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Utilizing Magnetic Electrospinning to Create Gradients in Fiber Alignment for Interfacial Tissue Engineering

Description

Heterogeneous tissues are composed of chemical and physical gradients responsible for transferring load from one tissue type to another, through the thickness or the length of the tissue. Musculoskeletal tissues

Heterogeneous tissues are composed of chemical and physical gradients responsible for transferring load from one tissue type to another, through the thickness or the length of the tissue. Musculoskeletal tissues include these junctions, such as the tendon-bone and ligament-bone, which consist of an alignment gradient through the length of the interfacial regions. These junctions are imperative for transferring mechanical loadings between dissimilar tissues. Engineering a proper scaffold that mimics the native architecture of these tissues to prompt proper repair after an interfacial injury has been difficult to fabricate within tissue engineering. Electrospinning is a common technique for fabricating nanofibrous scaffolds that can mimic the structure of the native extracellular matrix (ECM). However, current electrospinning techniques do not easily allow for the replication of the chemical and physical gradients present in musculoskeletal interfacial tissues. In this work, a novel magnetic electrospinning technique was developed to fabricate polycaprolactone (PCL) nanofibrous scaffolds that recapitulate the gradient alignment structure of the tendon-bone junction. When exposed to the natural magnetic field from a permanent magnet, PCL fibers innately aligned near the magnet with unalignment at distances further away from the magnetic field.

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Date Created
  • 2018-05

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Controlling the Electrospun Scaffold Profile at the Interface between Varying Fiber Alignment

Description

Musculoskeletal heterogenous tissues are crucial for dissipating mechanical load during physical activity. Modern procedures to repair these tissues have proven inadequate to restore full functionality, thus there is a need

Musculoskeletal heterogenous tissues are crucial for dissipating mechanical load during physical activity. Modern procedures to repair these tissues have proven inadequate to restore full functionality, thus there is a need for alternative reconstructive methods. Consequently, tissue engineered scaffolds can mimic the native structure of tissues and trigger a healing response. Heterogenous tissues like the tendon-bone junction consist of an interdigitated fiber alignment gradient from the tendon to the bone. It has been shown that electrospun fiber alignment gradients can be fabricated from the incorporation of magnetic fields. In this study, manipulating electrostatic and magnetic interactions from various electrospinning collector arrangements were investigated for creating an interdigitated fiber alignment gradient. The collector arrangement consisting of a magnet overlaid with razor cut aluminum foil proved to provide increased control over the interfacial shape. The rapid transition at the interfacial region was verified with brightfield microscopy revealing an interdigitated gradient from highly aligned fibers to unaligned fibers.

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  • 2020-05

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Characterization of Hyaluronic Acid Shear Thinning Hydrogels Towards Neural Cell Applications

Description

Traumatic brain injury (TBI) is a widespread health issue that affects approximately 1.7 million lives per year. The effects of TBI go past the incident of primary injury, as chronic

Traumatic brain injury (TBI) is a widespread health issue that affects approximately 1.7 million lives per year. The effects of TBI go past the incident of primary injury, as chronic damage can follow for years and cause irreversible neurodegeneration. A potential strategy for repair that has been studied is cell transplantation, as neural stem cells improve neurological function. While promising, neural stem cell transplantation presents challenges due to a relatively low survival rate post-implantation and issues with determining the optimal method of transplantation. Shear-thinning hydrogels are a type of hydrogel whose linkages break when under shear stress, exhibiting viscous flow, but reform and recover upon relaxation. Such properties allow them to be easily injected for minimally invasive delivery, while also shielding encapsulated cells from high shear forces, which would normally degrade the function and viability of such cells. As such, it is salient to research whether shear-thinning hydrogels are feasible candidates in neural cell transplantation applications for neuroregenerative medicine. In this honors thesis, shear-thinning hydrogels were formed through guest-host interactions of adamantane modified HA (guest ad-HA) and beta-cyclodextrin modified HA (host CD-HA). The purpose of the study was to characterize the injection force profile of different weight percentages of the HA shear-thinning hydrogel. The break force and average glide force were also compared between the differing weight percentages. By understanding the force exerted on the hydrogel when being injected, we could characterize how neural cells may respond to encapsulation and injection within HA shear-thinning hydrogels. We identified that 5% weight HA hydrogel required greater injection force than 4% weight HA hydrogel to be fully delivered. Such contexts are valuable, as this implies that higher weight percentage gels impart higher shear forces on encapsulated cells than lower weight gels. Further study is required to optimize our injection force system’s sensitivity and to investigate if cell encapsulation increases the force required for injection.

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  • 2021-05

Fabrication of Alignment and Chemical Gradient Scaffold for Tendon-Bone Repair using Electrospinning

Description

Heterogeneous musculoskeletal tissues, such as the tendon-bone junction, is crucial for transferring mechanical loading during human physical activity. This region, also known as the enthesis, is composed of a complex

Heterogeneous musculoskeletal tissues, such as the tendon-bone junction, is crucial for transferring mechanical loading during human physical activity. This region, also known as the enthesis, is composed of a complex extracellular matrix with gradient fiber orientations and chemistries. These different physical and chemical properties are crucial in providing the support that these junctions need in handling mechanical loading of everyday activities. Currently, surgical restorative procedures for a torn enthesis entail a very invasive technique of suturing the torn tendon onto the bone. This results in improper reinjury. To circumvent this issue, one common strategy within tissue engineering is to introduce a biomaterial scaffold which acts as a template for the local damaged tissue. Electrospinning can be utilized to fabricate a fibrous material to recapitulate the structure of the extracellular matrix. Currently electrospinning techniques only allow the creation of scaffold that consists of only one orientation and material. In this work, we investigated a multicomponent, magnetically assisted, electrospinning technique to fabricate a fiber alignment and chemical gradient scaffold for tendon-bone repair

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  • 2018-05

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Creating Biochemical Gradients via Photoconjugation and an In-House Designed Sliding Photomask

Description

Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds

Tissue engineering is an emerging field focused on the repair, replacement, and regeneration of damaged tissue. Engineered tissue consists of three factors: cells, biomolecular signals, and a scaffold. Cell-free scaffolds present a unique opportunity to develop highly specific microenvironments with tunable properties. Norbornene-functionalized hyaluronic acid (NorHA) hydrogels provide spatial control over biomolecule binding through a photopolymerization process. With this, biomimetic gradients can be produced to model a variety of tissue interfaces. To produce these patterns, a gradient mechanism was developed to function in tandem with a syringe pump. A conversion equation was derived to calculate a panel speed from the volumetric flow rate setting on the pump. Seven speeds were used to produce fluorophore gradients on the surface of NorHA hydrogels to assess changes in the length and slope of the gradient. The results indicated a strong positive linear correlation between the speed of the panel and the length of the gradient as well as a strong negative correlation between the speed of the panel and the slope of the gradient. Additionally, the mechanism was able to successfully produce several other types of gradients including multiregional, dual, and triregional.

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Date Created
  • 2019-05