Bryce M. Whited

Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds

Electrospun polymer/apatite composite scaffolds are promising candidates as functional bone substitutes because of their ability to allow pre-osteoblast attachment, proliferation, and differentiation. However these structures usually lack an adequate pore size to permit sufficient cell migration and colonization of the scaffold. To overcome this limitation, we developed an apatite-coated electrospun PLLA scaffold with varying pore size and porosity by utilizing a three-step water-soluble PEO fiber inclusion, dissolution, and mineralization process. The temporal and spatial dynamics of cell migration into the scaffolds were quantified to determine the effects of enhanced pore size and porosity on cell infiltration. MC3T3-E1 pre-osteoblast migration into the scaffolds was found to be a function of both initial PEO content and time. Scaffolds with greater initial PEO content (50% and 75% PEO) had drastically accelerated cell infiltration in addition to enhanced cell distribution throughout the scaffold when compared to scaffolds with lower PEO content (0% and 25% PEO). Furthermore, scaffolds with an apatite substrate significantly upregulated MC3T3-E1 alkaline phosphatase activity, osteocalcin content, and cell-mediated mineralization as compared to PLLA alone. These findings suggest that such a scaffold enhances pre-osteoblast infiltration, colonization, and maturation in vitro and may lead to overall improved bone formation when implanted in vivo.

The influence of electrospun scaffold topography on endothelial cell morphology, alignment, and adhesion in response to fluid flow

Bioengineered vascular grafts provide a promising alternative to autografts for replacing diseased or damaged arteries, but necessitate scaffold designs capable of supporting a confluent endothelium that resists endothelial cell (EC) detachment under fluid flow. To this end, we investigated whether tuning electrospun topography (i.e., fiber diameter and orientation) could impact EC morphology, alignment, and structural protein organization with the goal of forming a confluent and well‐adhered endothelium under fluid flow. To test this, a composite polymer blend of poly(ϵ‐caprolactone) (PCL) and type I collagen was electrospun to form scaffolds with controlled fiber diameters ranging from approximately 100–1,200 nm and with varying degrees of fiber alignment. ECs were seeded onto scaffolds, and cell morphology and degree of alignment were quantified using image analysis of fluorescently stained cells. Our results show that ECs form confluent monolayers on electrospun scaffolds, with cell alignment systematically increasing with a larger degree of fiber orientation. Additionally, cells on aligned electrospun scaffolds display thick F‐actin bundles parallel to the direction of fiber alignment and strong VE‐cadherin expression at cell–cell junctions. Under fluid flow, ECs on highly aligned scaffolds had greater resistance to detachment compared to cells cultured on randomly oriented and semi‐aligned scaffolds. These results indicate that scaffolds with aligned topographies may be useful in forming a confluent endothelium with enhanced EC adhesion for vascular tissue engineering applications. Biotechnol. Bioeng. 2014;111: 184–195.

Dynamic, Nondestructive Imaging of a Bioengineered Vascular Graft Endothelium

Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.

Imaging and characterization of bioengineered blood vessels within a bioreactor using free-space and catheter-based OCT

Regenerative medicine involves the bioengineering of a functional tissue or organ by seeding living cells on a biodegradable scaffold cultured in a bioreactor. A major barrier to creating functional tissues, however, has been the inability to monitor the dynamic and complex process of scaffold maturation in real time, making control and optimization extremely difficult. Current methods to assess maturation of bioengineered constructs, such as histology or organ bath physiology, are sample‐destructive. Optical coherence tomography (OCT) has recently emerged as a key modality for structural assessment of native blood vessels as well as engineered vessel mimics. The objective of this study was to monitor and assess in real time the development of a bioengineered blood vessel using a novel approach of combining both free‐space and catheter‐based OCT imaging in a new quartz‐walled bioreactor. Development of the blood vessel was characterized by changes in thickness and scattering coefficient over a 30‐day period.

Non-destructive real-time imaging of cell morphology for tissue-engineering applications

A major barrier for progress in regenerative medicine is the inability to observe the process of tissue regeneration non-destructively, in real time, and with cellular level resolution. In order to overcome this difficult challenge, we have proposed and developed an imaging-bioreactor system based on fiber micro-devices. Our system is based on directly incorporating multiple hollow core fibers (HCFs) into a biocompatible tissue scaffold. By inserting fiber-based micro-mirrors into the HCFs and applying a straightforward scanning-reconstruction algorithm, we have demonstrated an imaging resolution of ∼200 μm through an optically opaque scaffold.

Fabrication and characterization of poly(DL-lactic-co-glycolic acid)/zirconia-hybridized amorphous calcium phosphate composites.

Several minerals, such as hydroxyapatite and β-tricalcium phosphate, have been incorporated into bioresorbable polyester bone scaffolds to increase the osteoconductivity both in vitro and in vivo. More soluble forms of calcium phosphate that release calcium and phosphate ions have been postulated as factors that increase osteoblast differentiation and mineralization. Recently, a zirconia-hybridized pyrophosphate-stabilized amorphous calcium phosphate (Zr-ACP) has been synthesized allowing controlled release of calcium and phosphate ions. When incorporated into bioresorbable scaffolds, Zr-ACP has the potential to induce osteoconductivity. In this study, 80–90% (w/v) porous poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were formed by thermal phase separation from dioxane while incorporating Zr-ACP. Scanning electron microscopy revealed a highly porous structure with a pore size ranging from a few μm to about 100 μm, smaller than we had hoped for. Zr-ACP particles were evenly dispersed in the composite structure and incorporated into the pore walls. The amorphous structure of the Zr-ACP was maintained during composite fabrication, as found by X-ray diffraction. Composite scaffolds had larger compressive yield strengths and moduli compared to pure polymer scaffolds. These initial efforts demonstrate that PLGA/Zr-ACP composites can be formed in ways that ultimately serve as promising bone scaffolds in tissue engineering.

A Nondestructive Fiber-Based Imaging System to Assess Tissue-Engineered Vascular Grafts

The clinical need for alternatives to autologous vein and artery grafts for small-diameter vascular reconstruction have led researches to a tissue-engineering approach. Bioengineered vascular grafts provide a mechanically robust conduit for blood flow while implanted autologous cells remodel the construct to form a fully functional vessel [1]. A typical tissue-engineering approach involves fabricating a vascular scaffold from natural or synthetic materials, seeding the lumen of a vessel with endothelial cells (EC) and the vessel wall with smooth muscle cells or fibroblasts to mimic the functional properties of a native vessel. The cell-seeded vascular scaffold is then preconditioned in vitro using a pulsatile bioreactor to mimic in vivo conditions to enhance vessel maturation before implantation (Fig. 1).Copyright

Non-Destructive, Dynamic Imaging of HSP70 Response to Nanoparticle Mediated Photothermal Therapy in a 3D Tumor Mimic

Laser based photothermal therapy is a minimally invasive technique that relies on the absorption of energy by an irradiated tissue sample and results in the deposition of heat to destroy cancerous cells. The inclusion of nanoparticles that act as intense infrared absorbers allows for higher selectivity and additional absorption of laser energy into heat in the desired material. One promising carbonaceous nanoparticle is single walled carbon nanohorns (SWNHs) which have been demonstrated to be effective photoabsorbers [1].Copyright

Non-Destructive Real-Time Imaging of Cell Seeded Tissue Engineered Scaffolds

The use of tissue engineered scaffolds in combination with progenitor cells has emerged as a promising strategy to restore or replace tissues damaged by disease or trauma. In addition to being biocompatible and exhibiting appropriate mechanical properties, scaffolds must be designed to sustain cell attachment, proliferation, and differentiation to ultimately produce the desired tissue once implanted in the patient [1]. Conventional techniques used to assess successful scaffold design include cell viability stains, DNA assays, and histological sectioning/staining. While significant information can be gained from using these methodologies, they are destructive to the sample and only provide snapshots of scaffold and cell development at a limited number of time points. Consequently, key temporal and spatial information relating to tissue regeneration in the scaffold is lost utilizing these techniques. Thus, the ability to non-destructively monitor cell viability, proliferation, and differentiation in real-time is of great importance for scaffold design and tissue engineering [2].Copyright