John J. Lannutti

Compressive forces induce osteogenic gene expression in calvarial osteoblasts

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Youngs modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

Enhanced osteoblast response to a polymethylmethacrylate-hydroxyapatite composite.

Hydroxyapatite (HA)-reinforced polymers have been proposed as a method of improving the biological properties of bone cements and implant materials. For example, bone cements based on polymethylmethacrylate (PMMA) have long been used to secure orthopedic implants to the skeleton. This composite could also be used as a polished coating on other materials or in bulk form, shaped or molded, to custom fit a specific clinical need. However, complications may occur as a result of the limited mechanical and biological properties of PMMA. The purpose of this investigation was to determine whether the incorporation of HA in a PMMA matrix would enhance the biological properties of osteoblast response as compared to PMMA alone. Fetal rat calvarial osteoblasts were plated on discs of PMMA, PMMA/HA, commercially pure titanium (CpTi) and tissue culture polystyrene (control). Osteoblast attachment and day 2 proliferation were similar on all implant materials, whereas, day 8 proliferation on PMMA/HA was significantly higher than on PMMA and similar to CpTi and control. Extracellular matrix production was examined by immunohistochemistry which indicated that osteoblasts cultured on PMMA/HA showed a more distinct networked pattern of organized fibronectin. Histochemical staining of mineralization was examined by confocal microscopy which demonstrated a higher degree of mineralization in nodules formed on PMMA/HA as compared to PMMA. Together, these results indicate that the addition of HA in a PMMA matrix improves osteoblast response as compared to PMMA alone. Therefore, the incorporation of HA into a PMMA matrix may be a useful method to provide PMMA materials with enhanced osteogenic properties.

Measuring physical density with X-ray computed tomography

X-ray computed tomography (CT) can quantify variations in material density. However, little practical information concerning accurate translation of CT numbers (an approximation of the materials linear attenuation coefficient) into mass density exists. We describe our development of this methodology. Issues addressed include the physics of mapping CT density to mass density; development and scanning of density standards; post-reconstruction filtering to reduce the effects of quantum noise; and segmentation of the object data from the surrounding medium. Examples of our use of this procedure in studying density gradients during powder pressing and firing are given.

Micropatterning and characterization of electrospun poly(ε-caprolactone)/gelatin nanofiber tissue scaffolds by femtosecond laser ablation for tissue engineering applications.

Experimental investigations aimed at assessing the effectiveness of femtosecond (FS) laser ablation for creating microscale features on electrospun poly(ε‐caprolactone) (PCL)/gelatin nanofiber tissue scaffold capable of controlling cell distribution are described. Statistical comparisons of the fiber diameter and surface porosity on laser‐machined and as‐spun surface were made and results showed that laser ablation did not change the fiber surface morphology. The minimum feature size that could be created on electrospun nanofiber surfaces by direct‐write ablation was measured over a range of laser pulse energies. The minimum feature size that could be created was limited only by the pore size of the scaffold surface. The chemical states of PCL/gelatin nanofiber surfaces were measured before and after FS laser machining by attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy and X‐ray photoelectron spectroscopy (XPS) and showed that laser machining produced no changes in the chemistry of the surface. In vitro, mouse embryonic stem cells (mES cells) were cultured on as‐spun surfaces and in laser‐machined microwells. Cell densities were found to be statistically indistinguishable after 1 and 2 days of growth. Additionally, confocal microscope imaging confirmed that spreading of mES cells cultured within laser‐machined microwells was constrained by the cavity walls, the expected and desired function of these cavities. The geometric constraint caused statistically significant smaller density of cells in microwells after 3 days of growth. It was concluded that FS laser ablation is an effective process for microscale structuring of these electrospun nanofiber tissue scaffold surfaces. Biotechnol. Bioeng. 2011; 108:116–126.

Fabrication of burst pressure competent vascular grafts via electrospinning : Effects of microstructure

In this work, electrospun tubes of interest for vascular tissue engineering were fabricated and evaluated for burst pressure and suture retention strength (SRS) in the same context as tensile strength providing a direct, novel comparison. Tubes could be fabricated displaying average burst pressures up to 4000 mmHg--well above the standard of 2000 mmHg--and SRS values matching those of relevant natural tissues. Surprisingly, highly oriented fiber and maximal tensile properties are not absolutely necessary to attain clinically adequate burst pressures. The ability to resist bursting is clearly related to both initial solution solids loading and electrospinning deposition time. We make novel in situ observations of the relative microstructural characteristics of failure during bursting, and connect this to the conditions used to fabricate the graft. Processes typically thought to promote fiber alignment are, in fact, highly condition-dependent and do not always provide superior properties. In fact, electrospun structures displaying no discernable alignment could achieve burst pressures regarded clinically sufficient. The properties of individual electrospun fiber clearly do not fully dictate macroscale properties. Normal background levels of point bonding are enhanced by increased rotational speeds, and can have effects on properties more dominant than those of alignment.

Structuring electrospun polycaprolactone nanofiber tissue scaffolds by femtosecond laser ablation

Meshes of electrospun (ES) polycaprolactone (PCL) and polyethylene terephthalate nanofiber meshes were structured by ablation of linear grooves with a scanned femtosecond laser. Focus spot size, pulse energy, and scanning speed were varied to determine their affects on groove size and the characteristics of the electrospun fiber at the edges of these grooves. The femtosecond laser was seen to be an effective means for flexibly structuring the surface of ES PCL scaffolds. Femtosecond ablation resulted in much more uniformly ablated patterns compared to Q-switched nanosecond pulse laser ablation. Also, the width of the ablated grooves was well controlled by laser energy and focus spot size, although the grooves were significantly larger than the spot size. Also, some melting of fibers was observed at the edges of grooves. These affects were attributed to optical radiation from laser-induced plasma at higher pulse energies and melting of fibers at laser fluences lower than the ablation threshold. The ablatio...

Electrospun PCL in Vitro: a Microstructural Basis for Mechanical Property Changes

Polymeric tissue-engineering scaffolds must provide mechanical support while host-appropriate cells populate the structure and deposit extracellular matrix (ECM) components specific to the organ targeted for replacement. Even though this concept is widely shared, changes in polymer modulus and other mechanical properties versus biological exposure are largely unknown. This work shows that specific interactions of biological milieu with electrospun scaffolds can exert control over scaffold modulus. The net effects of biological and non-biological environments on electrospun structures following 7 and 28 days of in vitro exposure are established. Reduction of modulus, ultimate tensile strength and elongation occurs without the apparent involvement of classic hydrolysis mechanisms. We describe this phenomenon as deposition-induced inhibition of nanofiber rearrangement. This phenomenon shows that both mechanical and morphological characterization of electrospun structure under load in biological environments is required to tailor scaffold design to pursue specific tissue-engineering goals.

Hydrogel–electrospun fiber composite materials for hydrophilic protein release

Although hydrogels are widely used in controlled-release systems, obtaining extended, uniform drug release with little initial burst has been challenging. However, recently researchers have shown that combining hydrogels with another drug delivery material can dramatically improve release kinetics. Here we describe a novel hydrogel-based composite material that exhibits stable, near-linear, sustained release of a model hydrophilic protein (e.g., bovine albumin serum, BSA) for over two months with a significant reduction in initial burst release (7% vs. 20%). The composite is comprised of poly(ε-caprolactone) (PCL) electrospun fiber mats coupled with poly(ethylene glycol)-poly(ε-caprolactone) diacrylate (PEGPCL) hydrogels through photo-polymerization. It is believed that the additional diffusion barrier provided by hydrophobic electrospun fiber mats reduces hydrogel swelling and water penetration rates and increases the diffusion path length, resulting in delayed, more uniform drug release. Further, released proteins remain bioactive as demonstrated by PC12 cell neurite extension in response to released nerve growth factor (NGF). The use of electrospun fiber mats to modulate hydrogel drug release provides a new method to control release kinetics of hydrophilic proteins, reducing burst release and extending the release duration.

Structure-function relationships and source-to-ground distance in electrospun polycaprolactone.

The strength of electrospun scaffolds has direct relevance to their function within tissue engineering. We characterized the effects of source-to-ground distance on the mechanical properties of electrospun poly(epsilon-caprolactone) (PCL). Source-to-ground distances of 10, 15 and 20 cm, solids concentrations of 12 and 18 wt.% and mandrel rotation surface speeds of 0-12 m s(-1) were utilized. Tensile tests evaluated elastic modulus, tensile strength and elongation at failure. Scanning electron microscopy provided morphology and quantified fiber alignment. Increased source-to-ground distance yielded a microstructure allowing greater fiber rearrangement under load, tripling the observed tensile strength. Increases in rotational speed generally increased fiber alignment and strength at high but not low to moderate speeds. As fiber is quickly pulled out of a comparatively gentle falling process, collision with neighboring fibers moving at different speeds and in different directions can occur. The source-to-ground distance influences these collisions and thus has critical implications for microstructure and biocompatibility. In larger diameter (18 wt.% PCL), heavily point-bonded fibers (produced using a shorter, 10 cm source-to-ground distance), elongation at failure in the aligned direction increases dramatically due to severe localized necking. These specimens show only half of the tensile strength (from 2.6 to 4.5 MPa) and a dramatic increase (from 94% to 503%) in elongation at failure vs. a longer 20 cm source-to-ground distance. Strains of several hundred per cent are accompanied by periodic necking of large-diameter fibers in which microstructural failure appears to occur in a sequential manner involving an equilibrium between localized strain in the tensile direction and anisotropic point bonding that locally resists strain.

Effect of filler porosity on the abrasion resistance of nanoporous silica gel/polymer composites

OBJECTIVES This laboratory study was designed to investigate the effect of controlled nanoporosity on the wear resistance of polymeric composites reinforced with silica gel powders and to determine the mechanisms controlling the abrasive wear properties of these unique nanostructured materials. METHODS Silica gels were prepared by hydrolysis and condensation of tetraethylorthosilicate (TEOS) using four different catalysts to modify the porous structure of the resulting polysilicate silanation, an organic monomer (TEGDMA) containing various initiators was introduced into the gel powders to form a paste. The various pastes were then polymerized inside a glass mold. A pin-on-disk apparatus was then used to record the specimen length and number of revolutions. Abrasive wear rates were determined by regression analysis and statistical differences were determined by analysis of variance and multiple comparisons. BET was used to characterize the filler pore structure and scanning electron microscopy was used used to visually examine the abraded surfaces. RESULTS Significant differences (p < 0.05) in the wear rates of the experimental composites were noted. Within the range of filler porosities examined, wear resistance was found to be linearly dependent (R2 = 0.983) on filler pore volume. The wear rates decreased with increasing filler porosity. HCl-catalyzed gels having low porosity produced composites having relatively limited abrasion resistance. In contrast, high porosity HF-catalyzed gels produced more wear-resistant composites. The abrasive wear resistance of these nanocomposites was not significantly affected by the level of silane coupling used in these experiments. SEM evaluation suggested that better wear resistance was associated with fine-scale plastic deformation of the wear surface and the absence of filler particle pullout. SIGNIFICANCE Porous particles prepared via sol-gel show some promise as fillers that improve the wear resistance of photopolymerized resins. The wear resistance of the fillers appears to be directly related to nanoporous structure of the gel particles. Unlike conventional dental composites, these materials rely primarily on nanomechanical coupling for improved wear resistance. This new principle should benefit subsequent investigations.