Micromechanical Compatibility between Cells and Scaffolds Directs the Phenotypic Transition of Stem Cells.

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Thermal shrinkage reveals the feasibility of pulse-delay photocuring technique.

OBJECTIVES: To resolve the feasibility of the pulse-delay photocuring technique as a clinical strategy for reducing the detrimental polymerization stress induced in dental composites during the photocuring process. METHODS: Model dental composites with high and low-filler contents were cured with the pulse-delay photocuring technique using different combinations of photocuring variables (irradiance, exposure time, and delay time). Irradiance used ranged from 0.1W/cm2 to 4W/cm2. The exposure time of the first pulse varied from 0.2s to 27.2s and the delay times ranged from 10s to 120s. The radiant exposure was varied from 4J/cm2 to 20J/cm2. A cantilever-beam based instrument (NIST Standards Reference Instrument 6005) was used to implement the photocuring technique for the measurement of the polymerization properties (the degree of monomer conversion, polymerization stress induced due to shrinkage, and temperature change due to the reaction exotherm and curing light absorbance) simultaneously in real-time. These properties were compared with those obtained using the conventional photocuring technique (i.e., using a constant irradiance for a fixed exposure time, a uniform exposure). RESULTS: There exists a minimum radiant exposure, such that a reduction in the polymerization stress can be achieved without sacrificing the degree of monomer conversion by using the pulse-delay over the conventional photocuring technique. More specifically, stress reductions of up to 19% and 32% was observed with the pulse-delay when compared with the conventional photocuring technique at an irradiance of 0.5W/cm2 and 4W/cm2, respectively. The reduction occurred when the exposure time of the first pulse was greater than, but closer to, the gelation time (i.e., lower than the vitrification time) of the composite, regardless of the delay time used. Lower thermal shrinkage (contraction) during the post-curing time, rather than the stress relaxation during the delay time or lower degree of monomer conversion as claimed in the literature, is the cause of the reduction in the polymerization stress. SIGNIFICANCE: The study clarifies a long-standing confusion and controversy on the applicability of the pulse-delay photocuring technique for reducing the polymerization stress and promotes its potential clinical success for dental restorative composites.

Resin viscosity determines the condition for a valid exposure reciprocity law in dental composites.

OBJECTIVE: To provide conditions for the validity of the exposure reciprocity law as it pertains to the photopolymerization of dimethacrylate-based dental composites. METHODS: Composites made from different mass ratios of resin blends (Bis-GMA/TEGDMA and UDMA/TEGDMA) and silanized micro-sized glass fillers were used. All the composites used camphorquinone and ethyl 4-dimethylaminobenzoate as the photo initiator system. A cantilever beam-based instrument (NIST SRI 6005) coupled with NIR spectroscopy and a microprobe thermocouple was used to simultaneously measure the degree of conversion (DC), the polymerization stress (PS) due to the shrinkage, and the temperature change (TC) in real time during the photocuring process. The instrument has an integrated LED light curing unit providing irradiances ranging from 0.01W/cm2 to 4W/cm2 at a peak wavelength of 460nm (blue light). Vickers hardness of the composites was also measured. RESULTS: For every dental composite there exists a minimum radiant exposure required for an adequate polymerization (i.e., insignificant increase in polymerization with any further increase in the radiant exposure). This minimum predominantly depends on the resin viscosity of composite and can be predicted using an empirical equation established based on the test results. If the radiant exposure is above this minimum, the exposure reciprocity law is valid with respect to DC for high-fill composites (filler contents >50% by mass) while invalid for low-fill composites (that are clinically irrelevant). SIGNIFICANCE: The study promotes better understanding on the applicability of the exposure reciprocity law for dental composites. It also provides a guidance for altering the radiant exposure, with the clinically available curing light unit, needed to adequately cure the dental composite in question.

Bioinspired Wear-Resistant and Ultradurable Functional Gradient Coatings.

For mechanically protective coatings, the coating material usually requires sufficient stiffness and strength to resist external forces and meanwhile matched mechanical properties with the underneath substrate to maintain the structural integrity. These requirements generate a conflict that limits the coatings from achieving simultaneous surface properties (e.g., high wear-resistance) and coating/substrate interfacial durability. Herein this conflict is circumvented by developing a new manufacturing technique for functional gradient coatings (FGCs) with the material composition and mechanical properties gradually varying crossing the coating thickness. The FGC is realized by controlling the spatial distribution of magnetic-responsive nanoreinforcements inside a polymer matrix through a magnetic actuation process. By concentrating the reinforcements with hybrid sizes at the surface region and continuously diminishing toward the coating/substrate interface, the FGC is demonstrated to exhibit simultaneously high surface hardness, stiffness, and wear-resistance, as well as superb interfacial durability that outperforms the homogeneous counterparts over an order of magnitude. The concept of FGC represents a mechanically optimized strategy in achieving maximal performances with minimal use and site-specific distribution of the reinforcements, in accordance with the design principles of many load-bearing biological materials. The presented manufacturing technique for gradient nanocomposites can be extended to develop various bioinspired heterogeneous materials with desired mechanical performances.

Real-time synchronous measurement of curing characteristics and polymerization stress in bone cements with a cantilever-beam based instrument.

An instrumentation capable of simultaneously determining degree of conversion (DC), polymerization stress (PS), and polymerization exotherm (PE) in real time was introduced to self-curing bone cements. This comprises the combination of an in situ high-speed near-infrared spectrometer, a cantilever-beam instrument with compliance-variable feature, and a microprobe thermocouple. Two polymethylmethacrylate-based commercial bone cements, containing essentially the same raw materials but differ in their viscosity for orthopedic applications, were used to demonstrate the applicability of the instrumentation. The results show that for both the cements studied the final DC was marginally different, the final PS was different at the low compliance, the peak of the PE was similar, and their polymerization rates were significantly different. Systematic variation of instrumental compliance for testing reveals differences in the characteristics of PS profiles of both the cements. This emphasizes the importance of instrumental compliance in obtaining an accurate understanding of PS evaluation. Finally, the key advantage for the simultaneous measurements is that these polymerization properties can be correlated directly, thus providing higher measurement confidence and enables a more in-depth understanding of the network formation process.

The use of mechano growth factor to prevent cartilage degeneration in knee osteoarthritis.

Previous studies have attached more importance to growth factors in treating cartilage degeneration and osteoarthritis (OA). Here, the capability of mechano growth factor-C24E (MGF) to prevent osteoarthritic cartilage degeneration was evaluated in vitro and in vivo. Using in vitro cultured human OA chondrocytes treated with 10-60-ng/ml MGF for 12 hr, we detected the cell proliferation, migration, and anabolism of OA chondrocytes. The unfolded protein response and the protein characteristic of OA pathology, such as transforming growth factor ß, SMAD family member 3, and hypoxia-inducible factor 2α of OA chondrocytes, were also detected by western blotting. Furthermore, protein kinase RNA-like endoplasmic reticulum kinase was knocked down via small interfering RNA to illuminate the potential mechanism of MGF's treatment of OA. In a rabbit knee joint OA model, cartilage degeneration was inhibited after 2 weeks of treatment with 0.1-10-µg/ml MGF. This study demonstrated that MGF treatment can inhibit the pathological apoptosis of OA chondrocytes and promote the proliferation, migration, and matrix synthesis of the chondrocytes. The results also demonstrate that the degeneration of OA cartilage can be delayed by MGF treatment partially via unfolded protein response regulated by protein kinase RNA-like endoplasmic reticulum kinase and suggest a potential therapeutic application of MGF for OA treatment.

Quantifying the sensitivity of the network structure and properties from simultaneous measurements during photopolymerization.

We present a method that combines experimental and computational approaches to assess a comprehensive set of structural and functional evolution during a network formation process via photopolymerization. Our work uses the simultaneous measurement of the degree of conversion, polymerization stress, the change in reaction temperature, and shrinkage strain in situ. These measurements are combined with the theory of viscoelastic materials to deduce the relaxation time and frequency-dependent modulus of the polymerizing network. The relaxation time and degree of conversion are used to demonstrate the effect of processing parameters (e.g. curing protocol adjusted by the light intensity) in creating different network structures for the same initial resin. We describe experimental trends using effective medium calculations on a cross-linked polymer network model. In particular, we show that the effect of curing conditions on the spatial heterogeneity in crosslink density can be quantified using multiparametric measurements and modeling. Collectively, the present method is a way to examine holistically the complex structural and functional evolution of the network formation process.

Polymerization stress evolution of a bulk-fill flowable composite under different compliances.

OBJECTIVE: To use a compliance-variable instrument to simultaneously measure and compare the polymerization stress (PS) evolution, degree of conversion (DC), and exotherm of a bulk-fill flowable composite to a packable composite. METHODS: A bulk-fill flowable composite (Filtek Bulk-fill, FBF) and a conventional packable composite (Filtek Z250, Z250) purchased from 3M ESPE were investigated. The composites were studied using a cantilever-beam based instrument equipped with an in situ near infrared (NIR) spectrometer and a microprobe thermocouple. The measurements were carried out under various instrumental compliances (ranging from 0.3327µm/N to 12.3215µm/N) that are comparable to the compliances of clinically prepared tooth cavities. Correlations between the PS and temperature change as well as the DC were interpreted. RESULTS: The maximum PS of both composites at 10min after irradiation decreased with the increase in the compliance of the cantilever beam. The FBF composite generated a lower final stress than the Z250 sample under instrumental compliances less than ca. 4µm/N; however, both materials generated statistically similar PS values at higher compliances. The reaction exotherm and the DC of both materials were found to be independent of compliance. The DC of the FBF sample was slightly higher than that of the packable Z250 composite while the peak exotherm of FBF was almost double that of the Z250 composite. For FBF, a characteristic drop in the PS was observed during the early stage of polymerization for all compliances studied which was not observed in the Z250 sample. This drop was shown to relate to the greater exotherm of the less-filled FBF sample relative to the Z250 composite. SIGNIFICANCE: While the composites with lower filler content (low viscosity) are generally considered to have lower PS than the conventional packable composites, a bulk-fill flowable composite was shown to produce lower PS under a lower compliance of constraint as would be experienced if the composite was used as the base material in clinical procedures.

System compliance dictates the effect of composite filler content on polymerization shrinkage stress.

OBJECTIVE: The effect of filler content in dental restorative composites on the polymerization shrinkage stress (PS) is not straightforward and has caused much debate in the literature. Our objective in this study was to clarify the PS/filler content relationship based on analytical and experimental approaches, so that guidelines for materials comparison in terms of PS and clinical selection of dental composites with various filler content can be provided. METHODS: Analytically, a simplified model based on linear elasticity was utilized to predict PS as a function of filler content under various compliances of the testing system, a cantilever beam-based instrument used in this study. The predictions were validated by measuring PS of composites synthesized using 50/50 mixtures of two common dimethacrylate resins with a variety of filler contents. RESULTS: Both experiments and predictions indicated that the influence of filler content on the PS highly depended on the compliance of the testing system. Within the clinic-relevant range of compliances and for the specific sample configuration tested, the PS increased with increasing filler content at low compliance of instrument, while increasing the compliance caused the effect of filler content on the PS to gradually diminish. Eventually, at high compliance, the PS inverted and decreased with increasing filler content. SIGNIFICANCE: This compliance-dependent effect of filler content on PS suggests: (1) for materials comparison in terms of PS, the specific compliance at which the comparison being done should always be reported and (2) clinically, composites with relatively lower filler content could be selected for such cavities with relatively lower compliance (e.g. a Class-I cavity with thick tooth walls or the basal part in a cavity) and vice versa in order to reduce the final PS.

Regulation of BMP2-induced intracellular calcium increases in osteoblasts.

Although bone morphogenetic protein-2 (BMP2) is a well-characterized regulator that stimulates osteoblast differentiation, little is known about how it regulates intracellular Ca2+ signaling. In this study, intracellular Ca2+ concentration ([Ca2+ ]i ) upon BMP2 application, focal adhesion kinase (FAK) and Src activities were measured in the MC3T3-E1 osteoblast cell line using fluorescence resonance energy transfer-based biosensors. Increase in [Ca2+ ]i , FAK, and Src activities were observed during BMP2 stimulation. The removal of extracellular calcium, the application of membrane channel inhibitors streptomycin or nifedipine, the FAK inhibitor PF-573228 (PF228), and the alkaline phosphatase (ALP) siRNA all blocked the BMP2-stimulated [Ca2+ ]i increase, while the Src inhibitor PP1 did not. In contrast, a gentle decrease of endoplasmic reticulum calcium concentration was found after BMP2 stimulation, which could be blocked by both streptomycin and PP1. Further experiments revealed that BMP2-induced FAK activation could not be inhibited by PP1, ALP siRNA or the calcium channel inhibitor nifedipine. PF228, but not PP1 or calcium channel inhibitors, suppressed ALP elevation resulting from BMP2 stimulation. Therefore, our results suggest that BMP2 can increase [Ca2+ ]i through extracellular calcium influx regulated by FAK and ALP and can deplete ER calcium through Src signaling simultaneously. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1725-1733, 2016.

Correlation between polymerization shrinkage stress and C-factor depends upon cavity compliance.

OBJECTIVES: The literature reports inconsistent results regarding using configuration factor (C-factor) as an indicator to reflect the generation of polymerization shrinkage stress (PS) from dental restorative composites due to the constraint of cavity configuration. The current study aimed at unraveling the complex effects of C-factor on PS based on analytical and experimental approaches together, such that the reported inconsistency can be explained and a significance of C-factor in clinic can be comprehensively provided. METHODS: Analytical models based on linear elasticity were established to predict PS measured in instruments (testing systems) with different compliances, and complex effects of C-factor on PS were derived. The analyses were validated by experiments using a cantilever beam-based instrument and systematic variation of instrumental compliance. RESULTS: For a general trend, PS decreased with increasing C-factor when measured by instruments with high compliance. However, this trend gradually diminished and eventually reversed (PS became increased with increasing C-factor) by decreasing the system compliance. SIGNIFICANCE: Our study indicates that the correlation between PS and C-factor are highly dependent on the compliance of testing instrument for PS measurement. This suggests that the current concept on the role of C-factor in the stress development and transmission to tooth structures, higher C-factor produces higher PS due to reduced flow capacity of more confined materials, can be misleading. Thus, the compliance of the prepared tooth (cavity) structure should also be considered in the effect of C-factor on PS.

Perivascular-derived stem cells with neural crest characteristics are involved in tendon repair.

Tendons and ligaments exhibit limited regenerative capacity following injury, with damaged tissue being replaced by a fibrotic scar. The physiological role of scar tissue is complex and has been studied extensively. In this study, we demonstrate that rat tendons contain a unique subpopulation of cells exhibiting stem cell characteristics, including clonogenicity, multipotency, and self-renewal capacity. Additionally, these putative stem cells expressed markers consistent with neural crest stem cells (NCSCs). Using immunofluorescent labeling, we identified P75(+) (p75 neurotrophin receptor) cells in the perivascular regions of the native rat tendon. Importantly, P75(+) cells were frequently localized near vascular cells and increased in number within the peritenon after injury. Ultrastructural analysis showed that perivascular cells detached from vessels in response to injury, migrated into the interstitial space, and deposited extracellular matrix. Characterization of P75(+) cells isolated from the scar tissue indicated that this population also expressed the NCSC markers, Vimentin, Sox10, and Snail. In conclusion, our results suggest that neural crest-like stem cells of perivascular origin reside within the rat peritenon and give rise to scar-forming stromal cells following tendon injury.

Simultaneous measurement of polymerization stress and curing kinetics for photo-polymerized composites with high filler contents.

OBJECTIVES: Photopolymerized composites are used in a broad range of applications with their performance largely directed by reaction kinetics and contraction accompanying polymerization. The present study was to demonstrate an instrument capable of simultaneously collecting multiple kinetics parameters for a wide range of photopolymerizable systems: degree of conversion (DC), reaction exotherm, and polymerization stress (PS). METHODS: Our system consisted of a cantilever beam-based instrument (tensometer) that has been optimized to capture a large range of stress generated by lightly-filled to highly-filled composites. The sample configuration allows the tensometer to be coupled to a fast near infrared (NIR) spectrometer collecting spectra in transmission mode. RESULTS: Using our instrument design, simultaneous measurements of PS and DC are performed, for the first time, on a commercial composite with ≈80% (by mass) silica particle fillers. The in situ NIR spectrometer collects more than 10 spectra per second, allowing for thorough characterization of reaction kinetics. With increased instrument sensitivity coupled with the ability to collect real time reaction kinetics information, we show that the external constraint imposed by the cantilever beam during polymerization could affect the rate of cure and final degree of polymerization. SIGNIFICANCE: The present simultaneous measurement technique is expected to provide new insights into kinetics and property relationships for photopolymerized composites with high filler content such as dental restorative composites.

Relationships among cell morphology, intrinsic cell stiffness and cell-substrate interactions.

Cell modulus (stiffness) is a critical cell property that is important in normal cell functions and increasingly associated with disease states, yet most methods to characterize modulus may skew results. Here we show strong evidence indicating that the fundamental nature of free energies associated with cell/substrate interactions regulates adherent cell morphology and can be used to deduce cell modulus. These results are based on a mathematical model of biophysics and confirmed by the measured morphology of normal and cancerous liver cells adhered on a substrate. Cells select their final morphology by minimizing the total free energy in the cell/substrate system. The key mechanism by which substrate stiffness influences cell morphology is the energy tradeoff between the stabilizing influence of the cell-substrate interfacial adhesive energy and the destabilizing influence of the total elastic energies in the system. Using these findings, we establish a noninvasive methodology to determine the intrinsic modulus of cells by observing global changes in cell morphology in response to substrate stiffness. We also highlight the importance of selecting a relevant morphological index, cell roundness, that reflects the interchange between forms of energy governing cell morphology. Thus, cell-substrate interactions can be rationalized by the underlying biophysics, and cell modulus is easily measured.

Relative rigidity of cell-substrate effects on hepatic and hepatocellular carcinoma cell migration.

Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin ß1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin ß1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin ß1 expression.

Analyses of a cantilever-beam based instrument for evaluating the development of polymerization stresses.

OBJECTIVE: This investigation was to generate (1) guidelines for designing a tensometer that satisfies the necessary accuracy and sensitivity requirements for measuring polymerization stress (PS), and (2) a formula for calculating PS. Polymerization stress remains one of the most critical properties of polymeric dental materials, yet methods that can accurately quantify PS have been limited in part due to the complexity of polymerization, and in part due to the instrumentation itself. METHOD: In this study, we performed analytical and finite element analyses on a cantilever-beam based tensometer that is used to evaluate shrinkage stresses during the polymerization of dental restorative composites. RESULTS: The PS generated by a commercial dental composite determined using our new tensometer agrees with the predicted trend when the beam length and/or specimen height is varied. SIGNIFICANCE: This work demonstrates the importance of beam dimension and component relative rigidity to the accuracy of PS evaluation. An analytical solution is also derived for the vertical beam deflection, which can be used for any combination of bending and shearing to properly calculate the PS. In addition, an easy-to-conduct calibration procedure is provided that is desirable for periodic tensometer recalibration.

Solutions for determining equibiaxial substrate strain for dynamic cell culture.

In this work, empirical and analytical solutions of equibiaxial strain on a flexible substrate are derived for a dynamic cell culture system. The empirical formula, which fulfills the mechanistic conditions of the culture system, is based on a regression analysis from finite element analyses for a substrate undergoing large strains (<15%). The analytical (closed-form) solution is derived from the superposition of two elastic responses induced in the equibiaxial strain culture system after applying pressure to a substrate undergoing small strains (microstrains). There is good agreement between the strain predicted from the solutions and from the direct measurement. Using material and geometric properties of the culture system, the solutions developed here are straightforward and can be used to circumvent experimental measurements or finite element analysis to establish substrate pressure-strain relationships.

Local thickness and anisotropy approaches to characterize pore size distribution of three-dimensional porous networks.

Two image-analysis approaches for pore size distribution (PSD) of porous media are proposed. The methods are based on the skeleton representation of a porous object. One approach gives the local thickness of the pore object to represent the pore size corresponding to a lower limit of PSD. The other gives the pore size taking into account the anisotropy of pore object and corresponds to an upper limit of PSD. These two approaches can be incorporated into a computer program without computationally intensive and complex mathematical operations. In this study, these two approaches are applied to a two-dimensional (2D) synthetic image and 3D natural images of tissue scaffolds with various porosities and tortuosities. The scaffolds were prepared by removing the water-soluble poly(ethylene oxide) (PEO) component of the polycaprolactone (PCL)/PEO blend, leaving a porous PCL scaffold. Extracting quantitative PSD information for materials with an interconnected porous network rather than discrete voids (such as tissue scaffolds) is inevitably subjective without a universally accepted definition of "pore size." Therefore, the proposed lower and upper limits of PSD can come into play when considering mass transfer and scaffold surface area for cell-matrix interaction.

Cell morphology and migration linked to substrate rigidity.

A mathematical model, based on thermodynamics, was developed to demonstrate how substrate rigidity influences cell morphology and migration. The mechanisms by which substrate rigidity are translated into cell-morphological changes and cell movement are described. The model takes into account the competition between the elastic energies in the cell-substrate system and work of adhesion at the cell periphery. The cell morphology and migration are dictated by the minimum of the total free energy of the cell-substrate system. By using this model, reported experimental observations on cell morphological changes and migration can be better understood with a theoretical basis. In addition, these observations can be more accurately correlated with the variation of substrate rigidity. This study indicates that the activity of the adherent cell is dependent not only on the substrate rigidity but also is correlated with the relative rigidity between the cell and substrate. Moreover, the study suggests that the cell stiffness can be estimated based on the substrate stiffness corresponding to the change of trend in morphological stability.

Quantifying the directional parameter of structural anisotropy in porous media.

A new method has been developed to define the directional parameter and characterize the structural anisotropy of a highly porous structure with extensive pore interconnectivity and surface area, such as scaffolds in tissue engineering. This new method called intercept segment deviation (ISD) was validated through the comparison of structural anisotropy from ISD measurements with mechanical anisotropy from finite-element stress analysis. This was carried out on a generated two-dimensional (2D) image of a two-phase material and a real three-dimensional (3D) image of a tissue scaffold. The performance of other methods for quantification of the directional parameter was also assessed. The results indicate that the structural anisotropy obtained from this new method conforms to the actual mechanical anisotropy and provides a better prediction of the material orientation than the other methods for the 2D and 3D images studied.