Human Mesenchymal Cell Attachment Research Articles

Human Mesenchymal Cell Attachment, Growth and Biomineralization on Calcium-enriched Titania-polyester Coatings

Titanium implant osseointegration can be enhanced by surface modifications that include hydroxyapatite from Ca3(PO4)2. However, CaO may provide more surface calcium (w/w) to induce cellular responses. Therefore, the purpose of this study was to compare responses to novel CaO and Ca3(PO4)2-enriched titania-polyester (PPC) nanocomposite coatings, which were created by an electrostatic ultrafine dry powder coating technique. EDX confirmed the presence of a base polymer scaffold, biocompatible titanium, and CaO or Ca3(PO4)2. SEM showed that human embryonic palatal mesenchymal cells (ATCC CRL-1486) had attached and spread out onto all surfaces within 24 hours. Cell attachment assays showed that there was a progressive increase in cell numbers with surface CaO incorporation (0–5%), such that the PPC + 5% CaO coatings supported the most cells. Furthermore, the PPC + 5% CaO had significantly more (P = 0.006) cells attached to their surfaces than the PPC + 5% CaP coatings and titanium controls, at 24 hours. The PPC + 5% CaO also had more cells that had proliferated on their surfaces over 72 hours, although these differences were not significant (P > 0.05). Similarly, MTT assays showed that the cells had sustained metabolic activity on all surfaces. Again, metabolic activities were highest on the PPC + 5% CaO, and they were significantly higher (P < 0.05) on all CaO-enriched surfaces (1/3/5% CaO) than on the PPC + 5% CaP. Subsequently, Alizarin Red-S staining detected the initiation of biomineralization within 2 weeks, and abundant mineral deposits after 4 weeks of growth on PPC + 5% CaO and PPC + 3% CaO. These nanocomposite coatings have shown that CaO enrichments may provide a heightened cell response when compared to conventional hydroxyapatite.

Ultrafine calcium–titania–polyester dry powder coatings promote human mesenchymal cell attachment and biomineralization

An electrostatic ultrafine dry powder coating technique was developed, which utilizes nanoparticles to prevent agglomeration. When polyester resin, TiO2 (25%), nano-TiO2 (0.5%), filler, flow, degassing and curing agents were combined in a high-shear mixer, then sprayed (20kV) onto metal sheets and cured (200°C), biocompatible polymeric powder coatings (PPC) were created. This study's objective was to determine if these surface coatings could be successfully augmented with calcium-rich functional additives. Thus, formulations were enriched with CaO (1, 3 and 5%) and Ca3(PO4)2 (5%, CaP), and their coatings characterized. Particle size analysis of the powders confirmed their ultrafine dimensions. Volume diameters for 50% (D 0.5) were under 30μm, and 90% (D 0.9) under 50μm. Energy dispersive X-ray (EDX) analysis confirmed integration with minimal modification to the PPC base. Carbon (61–67%), oxygen (15–25%) and titanium (9–15%) dominated all surfaces, whereas calcium (0.5–1.0%) and phosphorus (0.3%) were only detected in the CaO and CaP enriched coatings respectively. Elemental mapping showed calcium clusters that were smaller but more abundant with more CaO (PPC+3%CaO, PPC+5%CaO), and colocalized to phosphorus in the PPC+5%CaP. Contact angle measurements indicated hydrophilic coatings (62–81°) and significantly (P<0.05) increased surface wetting with CaO. Optical microscopy showed human mesenchymal cell (HEPM, ATCC CRL-1486) attachment and spreading on all coatings within 24h, and increasing confluence with CaO incorporation. Alizarin Red-S staining detected the biomineralization of cultures on all coatings that increased with CaO and time. Ultimately, the PPC+5%CaO had the most cells and biomineralization. This ultrafine dry powder coating technology can create biocompatible surfaces from polyester resins and TiO2, which can be readily augmented with functional additives such as calcium, to titrate optimal cellular responses.

MTA-enriched nanocomposite TiO2-polymeric powder coatings support human mesenchymal cell attachment and growth

The objective of the study described in this paper was the development of novel polymer/ceramic nanocomposite coatings for implants through the application of ultrafine powder coating technology. Polyester resins were combined with µm-sized TiO2 (25%) as the biocompatibility agent, nTiO2 (0.5%) as the flow additive and mineral trioxide aggregates (ProRoot® MTA, 5%) as bioactive ceramics. Ultrafine powders were prepared and applied to titanium to create continuous polymeric powder coatings (PPCs) through the application of electrostatic ultrafine powder coating technology. Energy dispersive x-ray analysis confirmed that MTA had been incorporated into the PPCs, and elemental mapping showed that it had formed small clusters that were evenly distributed across the surface. Scanning electron microscopy (SEM) revealed continuous and smooth, but highly textured surface coatings that contrasted with the scalloped appearance of commercially pure titanium (cpTi) controls. Atomic force microscopy revealed intricate nano-topographies with an abundance of submicron-sized pits and nano-projections, evenly dispersed across their surfaces. Inverted fluorescence microscopy, SEM and cell counts showed that human embryonic palatal mesenchymal cells attached and spread out onto PPC and MTA-enriched PPCs within 24 h. Mitochondrial enzyme activity measured viable and metabolically active cells on all of the surfaces. After 72 h of growth, cell counts and metabolic activity were significantly higher (P < 0.05) on the grey-MTA enriched PPC surfaces, than on unmodified PPC and cpTi. The novel polymer/ceramic nanocomposites that were created with ultrafine powder coating technology were continuous, homogenous and nano-rough coatings that enhanced human mesenchymal cell attachment and growth.

Titania‐polymeric powder coatings with nano‐topography support enhanced human mesenchymal cell responses

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response.

TiO2-enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation

Novel polymeric powder coatings (PPC) were prepared by ultrafine powder coating technology and shown to support human mesenchymal cell attachment and growth. PPC surfaces enriched with nano-TiO2 (nTiO2) showed enhanced cellular responses, and were compared to commercially pure titanium (cpTi). After cell attachment and growth, osteogenic differentiation and bone matrix formation ensures osseointegration for implantable biomaterials. Therefore, the objective of this study was to determine if mesenchymal cells grown on PPC could undergo osteogenic differentiation by inducing Runx2 and bone matrix proteins, and then initiate mineralization. Atomic force microscopy revealed intricate three-dimensional micro-topographies, and the measures of nano-roughness and porosity were similar for all PPC surfaces. Scanning electron microscopy showed that the cells attached and spread out over all of the surfaces. After 1 week in osteogenic media, RT-PCR analysis showed the induction of Runx2, the up-regulation of type I collagen, and the initial detection of alkaline phosphatase and bone sialoprotein. After 4 weeks, Alizarin Red staining showed mineral deposition. However, cell spreading and osteogenic differentiation were significantly (P < 0.05) higher on the cpTi controls than on the PPC surfaces. Furthermore, spreading and differentiation were consistently higher on the titanium-enriched PPC-2, -3 and -4 than on the titanium-free PPC-1. Therefore, despite the presence of complex micro-topographies and nano-features, titanium-enrichment enhanced the cellular response, and pure titanium still provided the best substrate. These findings confirm the cytocompatibility of these novel polymeric coatings and suggest that titanium-enrichment and nTiO2 additives may enhance their performance.

Nano-TiO 2 Enriched Polymeric Powder Coatings Support Human Mesenchymal Cell Attachment and Growth

The objective of this study was to utilize ultrafine powder coating technology to prepare (PPC) that can support human mesenchymal cell attachment and growth. Resins were modified with titanium dioxide and polytetrafluoroethylene (PTFE), and enriched with either SiO(2) or TiO(2) nanoparticles (nSiO(2) or nTiO(2)) to create continuous PPC. Scanning electron microscopy (SEM) revealed complex surface topographies with nano features, and energy dispersive X-ray (EDX) analysis with Ti mapping confirmed a homogenous dispersion of the material. SEM and inverted fluorescence microscopy showed that human embryonic palatal mesenchymal (HEPM) cells attached and spread out on the PPC surfaces, particularly those enriched with nTiO( 2). Cell counts were higher, and the MTT assay measured more metabolic activity from the nTiO(2) enriched PPCs. Furthermore, these cellular responses were enhanced on PPC surfaces that were enriched with a higher concentration of nTiO(2) (2% vs. 0.5%), and appeared comparable to that seen on commercially pure titanium (cpTi). Therefore the nTiO( 2) enrichment of PPC was shown to favor human mesenchymal cell attachment and growth. Indeed, this modification of the materials created continuous surface coatings that sustained a favorable cellular response.

Chemical, modulus and cell attachment studies of reactive calcium phosphate filler-containing fast photo-curing, surface-degrading, polymeric bone adhesives

The initial structure, setting and degradation processes of a poly(lactide-co-propylene glycol-co-lactide) dimethacrylate adhesive filled with 50, 60 or 70 wt.% reactive calcium phosphates (monocalcium phosphate monohydrate (MCPM)/β-tricalcium phosphate (β-TCP)) have been assessed using nuclear magnetic resonance, Fourier transform infrared spectroscopy, Raman, X-ray powder diffraction and gravimetric studies. Filler incorporation reduced the rapid light-activated monomer polymerization rates slightly, but not the final levels. Upon immersion in water for 24 h, the set composite mass and volume increased due to water sorption. This promoted initial soluble MCPM loss from the composite surfaces, but also its reaction and monetite precipitation within the specimen bulk. After 48 h, composite gravimetric and chemical studies were consistent with surface erosion of polymer with reacted/remaining filler. The filled formulations exhibited more rapid early water sorption and subsequent surface erosion than the unfilled polymer. Calcium and phosphate release profiles and solution pH measurements confirmed early loss of surface MCPM with protons from polymer degradation products. At later times, the slower release of monetite/β-TCP buffered composite storage solutions at ∼5 instead of 3.2 for the unfilled polymer. Incorporation of filler increased both the early and later time material modulus. At intermediate times this effect was lost, presumably as a result of enhanced water sorption. The early modulus values obtained fell within the range reported for cancellous bone. Despite surface degradation, initial human mesenchymal cell attachment to both composites and polymer could be comparable with a non-degrading positive Thermanox® control. These studies indicate that the filled formulations may be good candidates for bone repair. Release of calcium and phosphate ions provides components essential for such repair.