Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electro-spinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.

Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen-mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.

A nanofibrous triphasic scaffold was electrospun from a mixture of polycaprolactone (PCL), type-I collagen and hydroxyapatite nanoparticles (nano-HA) with a mixture dry weight ratio of 50/30/20, respectively. Scaffolds were characterized by evaluating fiber morphology and chemical composition, dispersion of HA particles and nanoindentation. Scanning electron microscopy revealed fibers with an average diameter of 180 +/- 50 nm, which coincides well with the collagen fiber bundle diameter characteristic of the native extracellular matrix of bone. The triphasic fibers, stained with calcein and imaged with confocal microscopy, show a uniform dispersion of apatite particles throughout their length with minor agglomeration. Scaffold fibers of triphasic (50/30/20), collagen/nano-HA (80/20), PCL/nano-HA (80/20), pure PCL and pure collagen were each pressure consolidated into non-porous pellets for evaluation by transmission electron microscopy and nanoindentation. While the majority of apatite particles are uniformly dispersed having an average size of 30 nm, agglomerated particles as large as a few microns are sparsely distributed. Nanoindentation of the pressure-consolidated scaffolds showed a range of Young's modulus (0.50-3.9 GPa), with increasing average modulus in the order of (PCL < PCL/nano-HA < collagen < triphasic < collagen/nano-HA). The modulus data emphasize the importance of collagen and its interaction with other components in affecting mechanical properties of osteoconductive scaffolds.

Ultra-smooth nanostructured diamond (USND) can be applied to greatly increase the wear resistance of orthopaedic implants over conventional designs. Herein we describe surface modification techniques and cytocompatibility studies performed on this new material. We report that hydrogen (H)-terminated USND surfaces supported robust mesenchymal stem cell (MSC) adhesion and survival, while oxygen- (O) and fluorine (F)-terminated surfaces resisted cell adhesion, indicating that USND can be modified to either promote or prevent cell/biomaterial interactions. Given the favorable cell response to H-terminated USND, this material was further compared with two commonly used biocompatible metals, titanium alloy (Ti-6Al-4V) and cobalt chrome (CoCrMo). MSC adhesion and proliferation were significantly improved on USND compared with CoCrMo, although cell adhesion was greatest on Ti-6Al-4V. Comparable amounts of the pro-adhesive protein, fibronectin, were deposited from serum on the three substrates. Finally, MSCs were induced to undergo osteoblastic differentiation on the three materials, and deposition of a mineralized matrix was quantified. Similar amounts of mineral were deposited onto USND and CoCrMo, whereas mineral deposition was slightly higher on Ti-6Al-4V. When coupled with recently published wear studies, these in vitro results suggest that USND has the potential to reduce debris particle release from orthopaedic implants without compromising osseointegration.

Traditional cell culture substrates consist of static, flat surfaces although in vivo, cells exist on various dynamic topographies. We report development of a reconfigurable microtopographical system compatible with cell culture that is comprised of reversible wavy microfeatures on poly (dimethylsiloxane). Robust reversibility of the wavy micropattern is induced on the cell culture customized substrate by first plasma oxidizing the substrate to create a thin, brittle film on the surface and then applying and releasing compressive strain, to introduce and remove the microfeatures, respectively. The reversible topography was able to align, unalign, and realign C2C12 myogenic cell line cells repeatedly on the same substrate within 24 h intervals, and did not inhibit cell differentiation. The flexibility and simplicity of the materials and methods presented here provide a broadly applicable capability by which to investigate and compare dynamic cellular processes not yet easily studied using conventional in vitro culture substrates.