Chitosan-based composite bilayer scaffold as an in vitro osteochondral defect regeneration model.

Prolonged osteochondral tissue damage can result in osteoarthritis and decreased quality of life. Multiphasic scaffolds, where different layers model different microenvironments, are a promising treatment approach, yet stable joining between layers during fabrication remains challenging. Here, a bilayer scaffold for osteochondral tissue regeneration was fabricated using thermally-induced phase separation (TIPS). Two distinct polymer solutions were layered before TIPS, and the resulting porous, bilayer scaffold was characterized by seamless interfacial integration and a mechanical stiffness gradient reflecting the native osteochondral microenvironment. Chitosan is a critical component of both scaffold layers to facilitate cell attachment and the formation of polyelectrolyte complexes with other biologically relevant natural polymers. The articular cartilage region was optimized for hyaluronic acid content and stiffness, while the subchondral bone region was defined by higher stiffness and osteoconductive hydroxyapatite content. Following co-culture with chondrocyte-like (SW-1353 or mesenchymal stem cells) and osteoblast-like cells (MG63), cell proliferation and migration to the interface along with increased gene expression associated with relevant markers of osteogenesis and chondrogenesis indicates the potential of this bilayer scaffold for osteochondral tissue regeneration.

Fabrication and Characterization of Chitosan-Hyaluronic Acid Scaffolds with Varying Stiffness for Glioblastoma Cell Culture.

The invasive and recurrent nature of glioblastoma multiforme (GBM) is linked to a small subpopulation of cancer cells, which are self-renewing, resistant to standard treatment regimens, and induce formation of new tumors. Matrix stiffness is implicated in the regulation of cell proliferation, drug resistance, and reversion to a more invasive phenotype. Therefore, understanding the relationship between matrix stiffness and tumor cell behavior is vital to develop appropriate in vitro tumor models. Here, chitosan-hyaluronic acid (CHA) polyelectrolyte complex scaffolds are fabricated with statistically significant stiffness variances to characterize the effect of scaffold stiffness on morphology, proliferation, drug resistance, and gene expression in human glioblastoma cells (U-87 MG). All scaffolds support GBM proliferation over a 12-day culture period, yet larger spheroids are observed in scaffolds with higher stiffness. Additionally, GBM cells cultured in stiffer CHA scaffolds prove significantly more resistant to the common chemotherapeutic temozolomide. Moreover, the stiffer 8% CHA scaffolds exhibit an increase in expression of drug resistance and invasion related genes compared to 2D culture. CHA scaffolds present a tunable microenvironment for enhanced tumor cell malignancy and may provide a valuable in vitro microenvironment for studying tumor progression and screening anticancer therapies.

Modeling the tumor microenvironment using chitosan-alginate scaffolds to control the stem-like state of glioblastoma cells.

Better prediction of in vivo drug efficacy using in vitro models should greatly improve in vivo success. Here we utilize 3D highly porous chitosan-alginate complex scaffolds to probe how various components of the glioblastoma microenvironment including extracellular matrix and stromal cells affect tumor cell stem-like state.

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.

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.

Extending foldamer design beyond α-helix mimicry: α/ß-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 αâ†’ß replacements throughout this 19-mer sequence enabled us to identify homologues that contain up to ~30% ß residues, retain significant affinity for VEGF, and display substantial resistance to proteolysis. These α/ß-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 ß-TCP stability and enable growth factor binding and release.

ß-Tricalcium phosphate (ß-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 ß-TCP has a rapid and uncontrolled rate of degradation. In the current study ß-TCP granules were mineral coated with the aim of influencing the dissolution rate of ß-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 ß-TCP granules with mineral layers possessing different dissolution properties. The presence of a biomineral coating delays the dissolution rate of the ß-TCP granules. As the carbonate (CO(3)(2-)) content in the coating was increased the dissolution rate of the coated ß-TCP also increased, but remained slower than the dissolution of uncoated ß-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.

The effect of BMP-2 on micro- and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity.

It is well established that scaffolds for applications in bone tissue engineering require interconnected pores on the order of 100 microm for bone in growth and nutrient and waste transport. As a result, most studies have focused on scaffold macroporosity (>100 microm). More recently researchers have investigated the role of microporosity in calcium phosphate -based scaffolds. Osteointegration into macropores improves when scaffold rods or struts contain micropores, typically defined as pores less than approximately 50 microm. We recently demonstrated multiscale osteointegration, or growth into both macropores and intra-red micropores (<10 microm), of biphasic calcium phosphate (BCP) scaffolds. The combined effect of BMP-2, a potent osteoinductive growth factor, and multiscale porosity has yet to be investigated. In this study we implanted BCP scaffolds into porcine mandibular defects for 3, 6, 12 and 24 weeks and evaluated the effect of BMP-2 on multiscale osteointegration. The results showed that given this in vivo model BMP-2 influences osteointegration at the microscale, but not at the macroscale, but not at the macroscale. Cell density was higher in the rod micropores for scaffolds containing BMP-2 compared with controls at all time points, but BMP-2 was not required for bone formation in micropores. In contrast, there was essentially no difference in the fraction of bone in macropores for scaffolds with BMP-2 compared with controls. Additionally, bone in macropores seemed to have reached steady-state by 3 weeks. Multiscale osteointegration results in bone-scaffold composites that are fully osteointegrated, with no 'dead space'. These composites are likely to contain a continuous cell network as well as the potential for enhanced load transfer and improved mechanical properties.

Multiscale osteointegration as a new paradigm for the design of calcium phosphate scaffolds for bone regeneration.

The role of macropore size (>100 microm) and geometry in synthetic scaffolds for bone regeneration has been studied extensively, but successful translation to the clinic has been slow. Significantly less attention has been given to porosity at the microscale (0.5-10 microm). While some have shown that microporosity in calcium phosphate (CaP)-based scaffolds can improve rate and extent of bone formation in macropores, none has explored microporosity as an additional and important space for bone ingrowth. Here we show osteointegration of biphasic calcium phosphate (BCP) scaffolds at both the macro and micro length scales. Bone, osteoid, and osteogenic cells fill micropores in scaffold rods and osteocytes are embedded in mineralized matrix in micropores, without the addition of growth factors. This work further highlights the importance of considering design parameters at the microscale and demonstrates the possibility for a bone-scaffold composite with no "dead space." Embedded osteocytes distributed throughout microporous rods may form a mechanosensory network, which would not be possible in scaffolds without microporosity. Multiscale osteointegration has the potential to greatly improve overall performance of these scaffolds through an improvement of mechanical properties, load transfer, and stability in the long and short term, and represents a new paradigm for scaffold design.