Ibuprofen loaded PLA nanofibrous scaffolds increase proliferation of human skin cells in vitro and promote healing of full thickness incision wounds in vivo.

This article presents successful incorporation of ibuprofen in polylactic acid (PLA) nanofibers to create scaffolds for the treatment of both acute and chronic wounds. Nanofibrous PLA scaffolds containing 10, 20, or 30 wt % ibuprofen were created and ibuprofen release profiles quantified. In vitro cytotoxicity to human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) of the three scaffolds with varying ibuprofen concentrations were evaluated and compared to pure PLA nanofibrous scaffolds. Thereafter, scaffolds loaded with ibuprofen at the concentration that promoted human skin cell viability and proliferation (20 wt %) were evaluated in vivo in nude mice using a full thickness skin incision model to determine the ability of these scaffolds to promote skin regeneration and/or assist with scarless healing. Both acellular and HEK and HDF cell-seeded 20 wt % ibuprofen loaded nanofibrous bandages reduced wound contraction compared with wounds treated with Tegaderm™ and sterile gauze. Newly regenerated skin on wounds treated with cell-seeded 20 wt % ibuprofen bandages exhibited significantly greater blood vessel formation relative to acellular ibuprofen bandages. We have found that degradable anti-inflammatory scaffolds containing 20 wt % ibuprofen promote human skin cell viability and proliferation in vitro, reduce wound contraction in vivo, and when seeded with skin cells, also enhance new blood vessel formation. The approaches and results reported here hold promise for multiple skin tissue engineering and wound healing applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 327-339, 2017.

Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication of tissue engineering scaffolds.

Electrospun nonwovens have been used extensively for tissue engineering applications due to their inherent similarities with respect to fibre size and morphology to that of native extracellular matrix (ECM). However, fabrication of large scaffold constructs is time consuming, may require harsh organic solvents, and often results in mechanical properties inferior to the tissue being treated. In order to translate nonwoven based tissue engineering scaffold strategies to clinical use, a high throughput, repeatable, scalable, and economic manufacturing process is needed. We suggest that nonwoven industry standard high throughput manufacturing techniques (meltblowing, spunbond, and carding) can meet this need. In this study, meltblown, spunbond and carded poly(lactic acid) (PLA) nonwovens were evaluated as tissue engineering scaffolds using human adipose derived stem cells (hASC) and compared to electrospun nonwovens. Scaffolds were seeded with hASC and viability, proliferation, and differentiation were evaluated over the course of 3 weeks. We found that nonwovens manufactured via these industry standard, commercially relevant manufacturing techniques were capable of supporting hASC attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC, making them promising candidates for commercialization and translation of nonwoven scaffold based tissue engineering strategies.

Laser ablation imparts controlled micro-scale pores in electrospun scaffolds for tissue engineering applications.

Electrospun scaffolds have been used extensively for tissue engineering applications due to the simple processing scheme and versatility. However, many additional benefits can be imparted to these materials via post-processing techniques. Specifically the addition of structured pores on the micro-scale can offer a method to enable patterned cell adhesion, enhanced diffusional properties, and/or guide vascular infiltration upon implantation in vivo. In this study, we laser ablated electrospun poly(L: -lactic acid) (PLA) scaffolds and assessed the ablation process and cellular interaction by examining human adipose-derived stem cell (hASC) viability and proliferation on laser micro-machined scaffolds. Laser ablated pores of 150, 300, and 600 µm diameter were micro-machined through electrospun PLA scaffolds. Laser ablation parameters were varied and it was determined that the aperture and z-travel direction of the laser linearly correlated with the ablated pore diameter. To assess cytocompatibility of the micro-machined scaffolds, hASCs were seeded on each scaffold and cell viability was assessed on day 7. Human ASCs were able to adhere around the micro-machined features. DNA content was quantified on all scaffolds and it was determined that hASCs were able to proliferate on all scaffolds. The process of laser ablation could impart many beneficial features to electrospun scaffolds by increasing mass transport and mimicking micro-scale features and assisting in patterning of cells around micro-machined features.

Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells.

Development of tissue-engineered bone constructs has recently focused on the use of electrospun composite scaffolds seeded with stem cells from various source tissues. In this study, we fabricated electrospun composite scaffolds consisting of beta-tricalcium phosphate (TCP) crystals and poly(L-lactic acid) (PLA) at varying loading levels of TCP (0, 5, 10, 20 wt%) and assessed the composite scaffolds' material properties and ability to induce proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in the presence of osteogenic differentiating medium. The electrospun scaffolds all exhibited a nonwoven structure with an interconnected porous network. With the addition of TCP, the fiber diameter increased with each treatment ranging from 503.39 +/- 20.31 nm for 0 wt% TCP to 1267.36 +/- 59.03 nm for 20 wt% TCP. Tensile properties of the composite scaffolds were assessed and the overall tensile strength of the neat scaffold (0 wt% TCP) was 847 +/- 89.43 kPA; the addition of TCP significantly decreased this value to an average of 350.83 +/- 38.57 kPa. As the electrospun composite scaffolds degraded in vitro, TCP was released into the medium with the largest release occurring within the first 6 days. Human ASCs were able to adhere, proliferate and osteogenically differentiate on all scaffold combinations. DNA content increased in a temporal manner for each scaffold over 18 days in culture although for the day 12 timepoint, the 10 wt% TCP scaffold induced the greatest hASC proliferation. Endogenous alkaline phosphatase activity was enhanced on the composite PLA/TCP scaffolds compared to the PLA control particularly by day 18. It was noted that at the highest TCP loading levels of 10 and 20 wt%, there was a dramatic increase in the amount of cell-mediated mineralization compared to the 5 wt% TCP and the neat PLA scaffold. This work suggests that local environment cues provided by the biochemical nature of the scaffold can accelerate the overall osteogenic differentiation of hASCs and encourage rapid ossification.

Atomic layer deposition and biocompatibility of titanium nitride nano-coatings on cellulose fiber substrates.

Atomic layer deposition (ALD) is investigated as a process to produce inorganic metallic bio-adhesive coatings on cellulosic fiber substrates. The atomic layer deposition technique is known to be capable of forming highly conformal and uniform inorganic thin film coatings on a variety of complex surfaces, and this work presents an initial investigation of ALD on porous substrate materials to produce high-precision biocompatible titanium oxynitride coatings. X-ray photoelectron spectroscopy (XPS) confirmed TiNOx composition, and transmission electron microscopy (TEM) analysis showed the coatings to be uniform and conformal on the fiber surfaces. Biocompatibility of the modified structures was determined as a function of coating layer thickness by fluorescent live/dead staining of human adipose-derived adult stem cells (hADSC) at 6, 12 and 24 h. Cell adhesion showed that thin TiNOx coatings yielded the highest number of cells after 24 h with a sample coated with a 20 A coating having approximately 28.4 +/- 3.50 ng DNA. By altering the thickness of the deposited film, it was possible to control the amount of cells adhered to the samples. This work demonstrates the potential of low temperature ALD as a surface modification technique to produce biocompatible cellulose and other implant materials.

Monitored steady-state excitation and recovery (MSSER) radiation force imaging using viscoelastic models.

Acoustic radiation force imaging methods distinguish tissue structure and composition by monitoring tissue responses to applied radiation force excitations. Although these responses are a complex, multidimensional function of the geometric and viscoelastic nature of tissue, simplified discrete biomechanical models offer meaningful insight to the physical phenomena that govern induced tissue motion. Applying Voigt and standard linear viscoelastic tissue models, we present a new radiation force technique - monitored steady-state excitation and recovery (MSSER) imaging - that tracks both steady-state displacement during prolonged force application and transient response following force cessation to estimate tissue mechanical properties such as elasticity and viscosity. In concert with shear wave elasticity imaging (SWEI) estimates for Young's modulus, MSSER methods are useful for estimating tissue mechanical properties independent of the applied force magnitude. We test our methods in gelatin phantoms and excised pig muscle, with confirmation through mechanical property measurement. Our results measured 10.6 kPa, 14.7 kPa, and 17.1 kPa (gelatin) and 122.4 kPa (pig muscle) with less than 10% error. This work demonstrates the feasibility of MSSER imaging and merits further efforts to incorporate relevant mechanical tissue models into the development of novel radiation force imaging techniques.

Semiautomated finite element mesh generation methods for a long bone.

The objective of this work was to develop and test a semi-automated finite element mesh generation method using computed tomography (CT) image data of a canine radius. The present study employs a direct conversion from CT Hounsfield units to elastic moduli. Our method attempts to minimize user interaction and eliminate the need for mesh smoothing to produce a model suitable for finite element analysis. Validation of the computational model was conducted by loading the CT-imaged canine radius in four-point bending and using strain gages to record resultant strains that were then compared to strains calculated with the computational model. Geometry-based and uniform modulus voxel-based models were also constructed from the same imaging data set and compared. The nonuniform voxel-based model most accurately predicted the axial strain response of the sample bone (R(2)=0.9764).

Mechanobiology of soft skeletal tissue differentiation--a computational approach of a fiber-reinforced poroelastic model based on homogeneous and isotropic simplifications.

The material properties of multipotent mesenchymal tissue change dramatically during the differentiation process associated with skeletal regeneration. Using a mechanobiological tissue differentiation concept, and homogeneous and isotropic simplifications of a fiber-reinforced poroelastic model of soft skeletal tissues, we have developed a mathematical approach for describing time-dependent material property changes during the formation of cartilage, fibrocartilage, and fibrous tissue under various loading histories. In this approach, intermittently imposed fluid pressure and tensile strain regulate proteoglycan synthesis and collagen fibrillogenesis, assembly, cross-linking, and alignment to cause changes in tissue permeability (k), compressive aggregate modulus (H(A)), and tensile elastic modulus (E). In our isotropic model, k represents the permeability in the least permeable direction (perpendicular to the fibers) and E represents the tensile elastic modulus in the stiffest direction (parallel to the fibers). Cyclic fluid pressure causes an increase in proteoglycan synthesis, resulting in a decrease in k and increase in H(A) caused by the hydrophilic nature and large size of the aggregating proteoglycans. It further causes a slight increase in E owing to the stiffness added by newly synthesized type II collagen. Tensile strain increases the density, size, alignment, and cross-linking of collagen fibers thereby increasing E while also decreasing k as a result of an increased flow path length. The Poisson's ratio of the solid matrix, nu(s), is assumed to remain constant (near zero) for all soft tissues. Implementing a computer algorithm based on these concepts, we simulate progressive changes in material properties for differentiating tissues. Beginning with initial values of E=0.05 MPa, H(A)=0 MPa, and k=1 x 10(-13) m(4)/Ns for multipotent mesenchymal tissue, we predict final values of E=11 MPa, H(A)=1 MPa, and k=4.8 x 10(-15) m(4)/Ns for articular cartilage, E=339 MPa, H(A)=1 MPa, and k=9.5 x 10(-16) m(4)/Ns for fibrocartilage, and E=1,000 MPa, H(A)=0 MPa, and k=7.5 x 10(-16) m(4)/Ns for fibrous tissue. These final values are consistent with the values reported by other investigators and the time-dependent acquisition of these values is consistent with current knowledge of the differentiation process.

Mechanobiology of initial pseudarthrosis formation with oblique fractures.

Mechanical stresses play an important role in regulating tissue differentiation in a variety of contexts during skeletal development and regeneration. It has been shown that some intermittent loading at a fracture site can accelerate secondary fracture healing. However, it has not been shown how the stress and strain histories resulting from mechanical loading of a fracture might, in some cases, inhibit normal fracture healing and induce pseudarthrosis formation. In this study, finite element analysis is used to calculate hydrostatic stress and maximum principal tensile strain patterns in regenerating tissue around the site of an oblique fracture. Using a mechanobiologic view on tissue differentiation, we compared calculated stress and strain patterns within the fracture callus to the histomorphology of a typical oblique pseudarthrosis. Tissue differentiation predictions were consistent with the characteristic histomorphology of oblique pseudarthrosis: in the interfragmentary gap. tensile strains led to "cleavage" of the callus; at the ends of both fracture fragments, hydrostatic pressure and tensile strain caused fibrocartilage formation, and, at discrete locations of the periosteum at the oblique fracture ends, mild hydrostatic tension caused bone formation. We also found that discrete regions of high hydrostatic pressure correlated with locations of periosteal bone resorption. When previous findings with distraction osteogenesis are considered with these observations, it appears that low levels of hydrostatic pressure may be conducive to periosteal cartilage formation but high hydrostatic pressure may induce periosteal bone resorption during bone healing. We concluded that tissue differentiation in pseudarthrosis formation is consistent with concepts previously presented for understanding fracture healing, distraction osteogenesis, and joint formation.