Novel naturally crosslinked electrospun nanofibrous chitosan mats for guided bone regeneration membranes: material characterization and cytocompatibility.

Guided bone regeneration (GBR) barrier membranes are used to prevent soft tissue infiltration into the graft space during dental procedures that involve bone grafting. Chitosan materials have shown promise as GBR barrier membranes, due to their biocompatibility and predictable biodegradability, but degradation rates may still be too high for clinical applications. In this study, chitosan GBR membranes were electrospun using chitosan (70% deacetylated, 312 kDa, 5.5 w/v%), with or without the addition of 5 or 10 mm genipin, a natural crosslinking agent, in order to extend the degradation to meet the clinical target time frame of 4-6 months. Membranes were evaluated for fibre diameter, tensile strength, biodegradation rate, bond structure and cytocompatibility. Genipin addition, at 5 or 10 mm, resulted in median fibre diameters 184, 144 and 154 nm for uncrosslinked, 5 mm and 10 mm crosslinked, respectively. Crosslinking, examined by Fourier transform infrared spectroscopy, showed a decrease in N-H stretch as genipin levels were increased. Genipin-crosslinked mats exhibited only 22% degradation based on mass loss, as compared to 34% for uncrosslinked mats at 16 weeks in vitro. The ultimate tensile strength of the mats was increased by 165% to 32 MPa with 10 mm crosslinking as compared to the uncrosslinked mats. Finally, genipin-crosslinked mats supported the proliferation of SAOS-2 cells in a 5 day growth study, similar to uncrosslinked mats. These results suggest that electrospun chitosan mats may benefit from genipin crosslinking and have the potential to meet clinical degradation time frames for GBR applications.

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration.

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 electrospinning, 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.

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite.

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.

The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation, and on bone formation at hydroxyapatite surfaces.

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.

Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms.

The ST6Gal-I glycosyltransferase, which adds alpha2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased alpha2-6 sialylation of beta1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished alpha2-6 sialylation of integrins, inhibits cell adhesion to collagen I, a beta1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack beta1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by beta1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.

Mesenchymal stem cell interaction with ultra-smooth nanostructured diamond for wear-resistant orthopaedic implants.

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.

Reversible on-demand cell alignment using reconfigurable microtopography.

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.

Preliminary tribological evaluation of nanostructured diamond coatings against ultra-high molecular weight polyethylene.

BACKGROUND: Some loss of joint prostheses has been attributed to osteolytic loosening associated with debris from wear of polyethylene articulating against metal alloys. Reduced polyethylene wear has been reported with ceramics serving as an alternative counterface. METHODS: Nanostructured Diamond (NSD) coatings were deposited onto Ti6Al4V by microwave plasma-assisted chemical vapor deposition, with both hydrogen-rich (H-NSD) and helium-rich (He-NSD) feedgas mixtures. Pin-on-disk wear tests of polyethylene against NSD and CoCr were performed in serum lubrication at body temperature. Scanning electron microscopy was used to examine surface morphology, and nanoindentation was used to determine hardness and modulus of the polyethylene wear surfaces. Raman spectroscopy, surface roughness, and wettability analyses of the NSD coatings were performed. RESULTS: Raman spectroscopy confirmed sp(2) and sp(3) bonded carbon in the NSD coatings. No significant differences in wear factors were found between polyethylene on H-NSD, He-NSD, and CoCr, despite higher roughness and friction coefficients for the He-NSD and H-NSD coatings, compared with CoCr. Contact angles for the diamond coatings were reduced following the wear tests, indicating that these surfaces became more hydrophilic. Numerous small protuberances were observed on pins articulated against CoCr, and a single, large protuberance was observed in polyethylene-on-NSD. These features were conjectured to be reconsolidated polyethylene particles. Nanoindentation modulus and hardness of the worn polyethylene surfaces were lower for polyethylene-on-diamond than for polyethylene-on-CoCr. CONCLUSIONS: As a counterface to polyethylene, NSD-coated Ti6Al4V produced wear factors comparable to CoCr in the present pin-on-disk tests, a promising step towards its use in joint replacement bearing applications.

Mesenchymal stem cell adhesion and spreading on microwave plasma-nitrided titanium alloy.

Improved methods to increase surface hardness of metallic biomedical implants are being developed in an effort to minimize the formation of wear debris particles that cause local pain and inflammation. However, for many implant surface treatments, there is a risk of film delamination due to the mismatch of mechanical properties between the hard surface and the softer underlying metal. In this article, we describe the surface modification of titanium alloy (Ti-6Al-4V), using microwave plasma chemical vapor deposition to induce titanium nitride formation by nitrogen diffusion. The result is a gradual transition from a titanium nitride surface to the bulk titanium alloy, without a sharp interface that could otherwise lead to delamination. We demonstrate that vitronectin adsorption, as well as the adhesion and spreading of human mesenchymal stem cells to plasma-nitrided titanium is equivalent to that of Ti-6Al-4V, while hardness is improved 3- to 4-fold. These in vitro results suggest that the plasma nitriding technique has the potential to reduce wear, and the resulting debris particle release, of biomedical implants without compromising osseointegration; thus, minimizing the possibility of implant loosening over time.

Genomic organization of the channel catfish CD45 functional gene and CD45 pseudogenes.

CD45 is a transmembrane protein tyrosine phosphatase, which in mammals plays an important role in T and B cell receptor and cytokine signaling. Recently, a catfish cDNA was shown to contain all characteristic CD45 features: an alternatively spliced amino-terminus, a cysteine-rich region, three fibronectin domains, a transmembrane region, and two phosphotyrosine phosphatase domains. However, analyses of CD45 cDNAs from various catfish lymphoid cell lines demonstrated that catfish CD45 is unique in that it contains a large number of alternatively spliced exons. Sequence analyses of cDNAs derived from the catfish clonal B cell line 3B11 indicated that this cell line expresses up to 13 alternatively spliced exons. Furthermore, sequence similarity among the alternatively spliced exons suggested duplication events. To establish the exact number and organization of alternatively spliced exons, a bacterial artificial chromosome library was screened, and the catfish functional CD45 gene plus six CD45 pseudogenes were sequenced. The catfish functional CD45 gene spans 37 kb and contains 49 exons. In comparison, the human and pufferfish CD45 genes consist of 34 and 30 exons, respectively. This difference in the otherwise structurally conserved catfish gene is due to the presence of 18 alternatively spliced exons that were likely derived through several duplication events. In addition, duplication events were also likely involved in generating the six pseudogenes, truncated at the 3' ends. A similarly 3' truncated CD45 pseudogene is also present in the pufferfish genome, suggesting that this specific CD45 gene duplication occurred before catfish and pufferfish diverged (approximately 400 million years ago).

Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs.

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.