Quynh P. Pham

The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells.

The function and development of cells rely heavily on the signaling interactions with the surrounding extracellular matrix (ECM). Therefore, a tissue engineering scaffold should mimic native ECM to recreate the in vivo environment. Previously, we have shown that an in vitro generated ECM secreted by cultured cells enhances the mineralized matrix deposition of marrow stromal cells (MSCs). In this study, MSC expression of 45 bone-related genes using real-time reverse transcriptase polymerase chain reaction (RT-PCR) was determined. Upregulation of osteoblastic markers such as collagen type I, matrix extracellular phosphoglycoprotein with ASARM motif, parathyroid hormone receptor, and osteocalcin, indicated that the MSCs on plain titanium scaffolds differentiated down the osteoblastic lineage and deposited a mineralized matrix on day 12. Significant mineralized matrix deposition was observed as early as day 4 on ECM-containing scaffolds and was associated with the enhancement in expression of a subset of osteoblast-specific genes that included a 2-fold increase in osteopontin expression at day 1 and a 6.5-fold increase in osteocalcin expression at day 4 as well as downregulation of chondrogenic gene markers. These results were attributed to the cellular interactions with growth factors and matrix molecules that are likely present in the in vitro generated ECM since the genes for insulin-like growth factor 1, insulin-like growth factor 2, vascular endothelial growth factor, dentin matrix protein, collagen type IV, cartilage oligomeric protein, and matrix metalloproteinase 13 were significantly upregulated during ECM construct generation. Overall, the data demonstrate that modulation of MSC differentiation occurs at the transcriptional level and gene expression of bone-related proteins is differentially regulated by the ECM. This study presents enormous implications for tissue engineering strategies, as it demonstrates that modification of a biomaterial with an in vitro generated ECM containing cell-generated bioactive signaling molecules can effectively direct gene expression and differentiation of seeded progenitor cell populations.

Water-Soluble Fullerene (C60) Derivatives as Nonviral Gene-Delivery Vectors

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch−Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

The Role of Lipase and α-Amylase in the Degradation of Starch/Poly(ɛ-Caprolactone) Fiber Meshes and the Osteogenic Differentiation of Cultured Marrow Stromal Cells

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.

Analysis of the osteoinductive capacity and angiogenicity of an in vitro generated extracellular matrix.

In this study, the osteoinductive potential of an in vitro generated extracellular matrix (ECM) deposited by marrow stromal cells seeded onto titanium fiber mesh scaffolds and cultured in a flow perfusion bioreactor was investigated. Culture periods of 8, 12, and 16 days were selected to allow for different amounts of ECM deposition by the cells as well as ECM with varying degrees of maturity (Ti/ECM/d8, Ti/ECM/d12, and Ti/ECM/d16, respectively). These ECM-containing constructs were implanted intramuscularly in a rat animal model. After 56 days, histologic analysis of retrieved constructs revealed no bone formation in any of the implants. Surrounding many of the implants was a fibrous capsule, which was often interspersed with fat cells. Within the pore spaces, the predominant tissue response was the presence of blood vessels and young fibroblasts or fat cells. The number of blood vessels on a per area basis calculated from a histomorphometric analysis increased as a function of the amount of ECM within the implanted constructs, with a significant difference between Ti/ECM/d16 and plain Ti constructs. These results indicate that although an in vitro generated ECM alone may not induce bone formation at an ectopic site, its use may enhance the vascularization of implanted constructs.

In vivo bone biocompatibility and degradation of porous fumarate-based polymer/alumoxane nanocomposites for bone tissue engineering.

The objective of this study was to determine how the incorporation of surface-modified alumoxane nanoparticles into a biodegradable fumarate-based polymer affects in vivo bone biocompatibility (characterized by direct bone contact and bone ingrowth) and in vivo degradability. Porous scaffolds were fabricated from four materials: poly(propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA) polymer alone; a macrocomposite consisting of PPF/PF-DA polymer with boehmite microparticles; a nanocomposite composed of PPF/PF-DA polymer and mechanically reinforcing surface-modified alumoxane nanoparticles; and a low-molecular weight PPF polymer alone (tested as a degradation control). Scaffolds were implanted in the lateral femoral condyle of adult goats for 12 weeks and evaluated by micro-computed tomography and histological analysis. For all material groups, small amounts of bone, some soft tissue, and a few inflammatory elements were observed within the pores of scaffolds, though many pores remained empty or filled with fluid only. Direct contact between scaffolds and surrounding bone tissue was also observed in all scaffold types, though less commonly. Minimal in vivo degradation occurred during the 12 weeks of implantation in all materials except the degradation control. These results demonstrate that the incorporation of alumoxane nanoparticles into porous PPF/PF-DA scaffolds does not significantly alter in vivo bone biocompatibility or degradation.

Natural stimulus responsive scaffolds/cells for bone tissue engineering: influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings.

This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g/L) showed the formation of pores, indicating an increase of porosity ( approximately 5-55% up to 21 days) resulting in porous three-dimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.

Slit-Surface Electrospinning: A Novel Process Developed for High-Throughput Fabrication of Core-Sheath Fibers

In this work, we report on the development of slit-surface electrospinning – a process that co-localizes two solutions along a slit surface to spontaneously emit multiple core-sheath cone-jets at rates of up to 1 L/h. To the best of our knowledge, this is the first time that production of electrospun core-sheath fibers has been scaled to this magnitude. Fibers produced in this study were defect-free (i.e. non-beaded) and core-sheath geometry was visually confirmed under scanning electron microscopy. The versatility of our system was demonstrated by fabrication of (1) fibers encapsulating a drug, (2) bicomponent fibers, (3) hollow fibers, and (4) fibers from a polymer that is not normally electrospinnable. Additionally, we demonstrate control of the process by modulating parameters such as flow rate, solution viscosity, and fixture design. The technological achievements demonstrated in this work significantly advance core-sheath electrospinning towards commercial and manufacturing viability.

The development of bioresorbable composite polymeric implants with high mechanical strength

Implants for the treatment of tissue defects should mimic the mechanical properties of the native tissue of interest and should be resorbable as well as biocompatible. In this work, we developed a scaffold from variants of poly(glycolic) acid which were braided and coated with an elastomer of poly(glycolide-co-caprolactone) and crosslinked. The coating of the scaffold with the elastomer led to higher mechanical strength in terms of compression, expansion and elasticity compared to braids without the elastomer coating. These composite scaffolds were found to have expansion properties similar to metallic stents, utilizing materials which are typically much weaker than metal. We optimized the mechanical properties of the implant by tuning the elastomer branching structure, crosslink density, and molecular weight. The scaffolds were shown to be highly resorbable following implantation in a porcine femoral artery. Biocompatibility was studied in vivo in an ovine model by implanting the scaffolds into femoral arteries. The scaffolds were able to support an expanded open lumen over 12 months in vivo and also fully resorbed by 18 months in the ovine model.