Sheeny K. Lan Levengood

Extending Foldamer Design beyond α-Helix Mimicry: α/β-Peptide Inhibitors of Vascular Endothelial Growth Factor Signaling

Diverse strategies have been explored to mimic the surface displayed by an α-helical segment of a protein, with the goal of creating inhibitors of helix-mediated protein-protein interactions. Many recognition surfaces on proteins, however, are topologically more complex and less regular than a single α-helix. We describe efforts to develop peptidic foldamers that bind to the irregular receptor-recognition surface of vascular endothelial growth factor (VEGF). Our approach begins with a 19-residue α-peptide previously reported by Fairbrother et al. (Biochemistry 1998, 37, 17754) to bind to this surface on VEGF. Systematic evaluation of α→β replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ~30% β residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/β-peptides can block VEGF-stimulated proliferation of human umbilical vein endothelial cells.

Specific VEGF sequestering and release using peptide-functionalized hydrogel microspheres

Growth factor signaling plays an essential role in regulating processes such as tissue development, maintenance, and repair. Gene expression levels, diffusion, degradation, and sequestration by extracellular matrix components all play a role in regulating the concentration of growth factors within the cellular microenvironment. Herein, we describe the synthesis and characterization of hydrogel microspheres that mimic the ability of the native extracellular matrix to reversibly bind vascular endothelial growth factor (VEGF) out of solution. A peptide ligand derived from the VEGF receptor 2 (VEGFR2) was covalently incorporated into the hydrogel microspheres in order to achieve binding affinity and specificity. In addition to being able to both bind and release VEGF in a controllable manner, the microspheres were also shown to affect human umbilical vein endothelial cell (HUVEC) proliferation. The resulting microspheres may enable new strategies to specifically upregulate or downregulate growth factor signaling in the cellular microenvironment.

Mineral coatings modulate β-TCP stability and enable growth factor binding and release.

β-Tricalcium phosphate (β-TCP) is an attractive ceramic for bone tissue repair because of its similar composition to bone mineral and its osteoconductivity. However, compared with other ceramics β-TCP has a rapid and uncontrolled rate of degradation. In the current study β-TCP granules were mineral coated with the aim of influencing the dissolution rate of β-TCP, and also to use the coating as a carrier for controlled release of the growth factors recombinant human vascular endothelial growth factor (rhVEGF), modular VEGF peptide (mVEGF), and modular bone morphogenetic protein 2 peptide (mBMP2). The biomineral coatings were formed by heterogeneous nucleation in aqueous solution using simulated body fluid solutions with varying concentrations of bicarbonate (HCO(3)). Our results demonstrate that we could coat β-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the β-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated β-TCP also increased, but remained slower than the dissolution of uncoated β-TCP. In addition, we showed sustained release of multiple growth factors, with release kinetics that were controllable by varying the identity of the growth factor or the CO(3)(2-) content in the mineral coating. Released rhVEGF induced human umbilical vein endothelial cell (HUVEC) proliferation, and mVEGF enhanced migration of mouse embryonic endothelial cells in a scratch wound healing assay, indicating that each released growth factor was biologically active.

Human endothelial colony forming cells undergo vasculogenesis within biphasic calcium phosphate bone tissue engineering constructs

An important consideration in bone regeneration is the need for expedited neovascularization within the defect site. Formation of a vascular network is critical for cell viability and normal function leading to tissue regeneration, but spontaneous angiogenesis is too slow to yield sufficient vessel formation. In this pilot study, human umbilical cord blood (hUCB)-derived endothelial colony forming cells (ECFCs) were evaluated for in vivo vasculogenesis in the macropores of biphasic calcium phosphate (BCP)/bone morphogenetic protein-2 (BMP-2) bone tissue engineering constructs. Constructs were implanted on the abdominal wall of NOD/SCID mice for 4 weeks. This study demonstrated in vivo vasculogenesis by human ECFCs within the macropore space of BCP/BMP-2 constructs. The human ECFC-derived vessels anastomosed with the host vasculature and perfused vessels were visible in the very center of the 5mm diameter, 2.5mm tall scaffolds. Additionally, the vessels were evenly distributed throughout the construct. This study suggests that scaffolds containing ECFCs have significant potential for expedited neovascularization in bony defects.

High affinity binding of an engineered, modular peptide to bone tissue.

Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

Automated segmentation of micro-CT images of bone formation in calcium phosphate scaffolds

In this work, we develop and validate an automated micro-computed tomography (micro-CT) image segmentation algorithm that accurately and efficiently segments bone, calcium phosphate (CaP)-based bone scaffold, and soft tissue. The algorithm enables quantitative evaluation of bone growth in CaP scaffolds in our study that includes many samples (100+) and large data sets (900 images per sample). The use of micro-CT for such applications is otherwise limited because the similarity in X-ray attenuation for the two materials makes them indistinguishable. Destructive characterization using histological techniques and scanning electron microscopy (SEM) has been the standard for CaP scaffolds, but these methods are cumbersome, inaccurate, and yield only 2D information. The proposed algorithm exploits scaffold periodicity and combines signal analysis, edge detection, and knowledge of three-dimensional spatial relationships between bone, CaP scaffold, and soft tissue to achieve fast and accurate segmentation. Application of this algorithm can lead to a new understanding of the role of CaP and scaffold internal structure on patterns and rates of bone growth.

Regulating Specific Growth Factor Signaling Using Immobilized Branched Ligands

VEGF-binding peptide ligands are incorporated into hydrogel microspheres and reduce the amount of growth factor in solution. VEGF binding affinity is enhanced by creating ligands with a dimer structure. The spheres are able to knock down VEGF-mediated HUVEC growth and reduce calcium signaling. The binding interaction is reversible, allowing the spheres to be used as a VEGF delivery vehicle.

Biomaterials for high-throughput stem cell culture.

A cells microenvironment plays a primary role in defining cell fate during tissue development, physiological function, and pathological dysfunction. Understanding the key components and interactions within these microenvironments is critical for effective use of stem cells for disease modeling and therapeutic applications. Yet cell microenvironments are difficult to study, as there are tens or hundreds of parameters that can influence cell behavior simultaneously. Additionally, parameters such as cell-cell interactions, cell-ECM interactions, cell shape, soluble signals, and mechanical forces vary dynamically in 3-dimensional space and time. The number of relevant experimental conditions in these cell-based biological systems quickly becomes intractable using standard experimental platforms and techniques. A new set of strategies involving high-throughput experimental formats and 3-dimensional culture is required to achieve significant progress in understanding and exploiting stem cell biology. This mini-review describes bioengineering approaches that are enabling for high-throughput stem cell culture, screening and analysis.