Determine the vertical ground reaction forces and knee mechanics with different gait inclinations in the sheep model.

Our current understanding of knee mechanics and anterior cruciate ligament (ACL) function is predominately based on data recorded during simulations of clinical examinations or the application of nonphysiologic loads and motions. These methodologies provide little information on knee and ACL mechanics during activities of daily living (ADLs). Additionally, researchers have not directly measured knee kinetics, knee contact pressures, and ACL forces, and it is unknown how these parameters change with different activities. This study quantified the effects of activity level on vertical ground reaction forces, knee kinematics, and joint and ligament forces during in vivo motions. Five female Suffolk sheep were walked twice weekly on a treadmill during level (0°), inclined (+6°), and declined (-6°) gait for 12 weeks. Electromagnetic (EM) trackers were surgically implanted onto the left distal femur and the left proximal tibia, and in vivo motions were recorded for all activities. Following sacrifice, the in vivo motions were applied to their respective knees using a serial robot with a multi-axis load cell. In vitro simulations were repeated to measure (a) total knee forces, (b) contact pressure maps, and (c) ACL-only forces. Declining the gait surface led to increased posterior translation during the swing phase and decreased flexion at hoof-strike, decreased medial contact pressure at push-off, decreased ACL force at hoof-strike and increased ACL force at push-off. This study established a system that can be used to examine knee mechanics and ACL forces during ADLs for different knee states to define design requirements for ACL reconstruction techniques.

Distortion in time perception as a result of concern about appearing biased.

Two experiments illustrate that the perception of a given time duration slows when white participants observe faces of black men, but only if participants are concerned with appearing biased. In Experiment 1 the concern with the appearance of bias is measured as a chronic state using the external motivation to respond without prejudice scale (Plant & Devine, 1998). In Experiment 2 it is manipulated by varying the race of the experimenter (black versus white). Time perception is assessed via a temporal discrimination task commonly used in the literature. Models of time perception identify arousal as a factor that causes perceived time to slow, and we speculate that arousal arising in intergroup interactions can alter time perception.

The LG/J murine strain exhibits near-normal tendon biomechanical properties following a full-length central patellar tendon defect.

PURPOSE OF THE STUDY: Identifying biological success criteria is needed to improve therapies, and one strategy for identifying them is to analyze the RNA transcriptome for successful and unsuccessful models of tendon healing. We have characterized the MRL/MpJ murine strain and found improved mechanical outcomes following a central patellar tendon (PT) injury. In this study, we evaluate the healing of the LG/J murine strain, which comprises 75% of the MRL/MpJ background, to determine if the LG/J also exhibits improved biomechanical properties following injury and to determine differentially expressed transcription factors across the C57BL/6, MRL/MpJ and the LG/J strains during the early stages of healing. MATERIALS AND METHODS: A full-length, central PT defect was created in 16-20 week old MRL/MpJ, LG/J, and C57BL/6 murine strains. Mechanical properties were assessed at 2, 5, and 8 weeks post surgery. Transcriptomic expression was assessed at 3, 7, and 14 days following injury using a novel clustering software program to evaluate differential expression of transcription factors. RESULTS: Average LG/J structural properties improved to 96.7% and 97.2% of native LG/J PT stiffness and ultimate load by 8 weeks post surgery, respectively. We found the LG/J responded by increasing expression of transcription factors implicated in the inflammatory response and collagen fibril organization. CONCLUSIONS: The LG/J strain returns to normal structural properties by 8 weeks, with steadily increasing properties at each time point. Future work will characterize the cell populations responding to injury and investigate the role of the differentially expressed transcription factors during healing.

On Race and Time.

Arousal is known to shape time perception, and heightened arousal causes one to perceive that time has slowed (i.e., a given length of time feels longer than it actually is). The current experiments illustrate that among White people who experience arousal when contemplating race (specifically those for whom appearing biased is an ongoing concern), time perception slows when they observe faces of Black men. We asked participants to judge the duration of presentation for faces of White and Black men (shown for periods ranging from 300 to 1,200 ms) relative to a standard duration of 600 ms. Evidence of bias emerged when White participants concerned with bias saw faces of Black men (e.g., durations of less than 600 ms were perceived as being greater than 600 ms). The current findings have implications for intergroup interactions in which timing is essential-for example, length of job interviews, police officers' perception of the length of an encounter and when force should be initiated, and doctors' perception of the length of medical encounters. Racially biased time perception is a new form of implicit bias, one exerted at the perceptual level.

Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury.

Musculoskeletal injuries greatly affect the U.S. population and current clinical approaches fail to restore long-term native tissue structure and function. Tissue engineering is a strategy advocated to improve tendon healing; however, the field still needs to establish biological benchmarks for assessing the effectiveness of tissue-engineered structures. Investigating superior healing models, such as the MRL/MpJ, offers the opportunity to first characterize successful healing and then apply experimental findings to tissue-engineered therapies. This study seeks to evaluate the MRL/MpJ's healing response following a central patellar tendon injury compared to wildtype. Gene expression and histology were assessed at 3, 7, and 14 days following injury and mechanical properties were measured at 2, 5, and 8 weeks. Native patellar tendon biological and mechanical properties were not different between strains. Following injury, the MRL/MpJ displayed increased mechanical properties between 5 and 8 weeks; however, early tenogenic expression patterns were not different between the strains. Furthermore, expression of the cyclin-dependent kinase inhibitor, p21, was not different between strains, suggesting an alternative mechanism may be driving the healing response. Future studies will investigate collagen structure and alignment of the repair tissue and characterize the complete healing transcriptome to identify mechanisms driving the MRL/MpJ response.

Functional tissue engineering of tendon: Establishing biological success criteria for improving tendon repair.

Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.

Evolving strategies in mechanobiology to more effectively treat damaged musculoskeletal tissues.

In this paper, we had four primary objectives. (1) We reviewed a brief history of the Lissner award and the individual for whom it is named, H.R. Lissner. We examined the type (musculoskeletal, cardiovascular, and other) and scale (organism to molecular) of research performed by prior Lissner awardees using a hierarchical paradigm adopted at the 2007 Biomechanics Summit of the US National Committee on Biomechanics. (2) We compared the research conducted by the Lissner award winners working in the musculoskeletal (MS) field with the evolution of our MS research and showed similar trends in scale over the past 35 years. (3) We discussed our evolving mechanobiology strategies for treating musculoskeletal injuries by accounting for clinical, biomechanical, and biological considerations. These strategies included studies to determine the function of the anterior cruciate ligament and its graft replacements as well as novel methods to enhance soft tissue healing using tissue engineering, functional tissue engineering, and, more recently, fundamental tissue engineering approaches. (4) We concluded with thoughts about future directions, suggesting grand challenges still facing bioengineers as well as the immense opportunities for young investigators working in musculoskeletal research. Hopefully, these retrospective and prospective analyses will be useful as the ASME Bioengineering Division charts future research directions.

The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon.

Tendon injuries are major orthopedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development.

Temporal discrimination of sub- and suprasecond time intervals: a voxel-based lesion mapping analysis.

We used voxel-based lesion-symptom mapping (VLSM) to determine which brain areas are necessary for discriminating time intervals above and below 1 s. VLSM compares behavioral scores of patients that have damage to a given voxel to those that do not on a voxel-by-voxel basis to determine which voxels are critical for the given behavior. Forty-seven subjects with unilateral hemispheric lesions performed a temporal discrimination task in which a standard stimulus was compared on each trial to a test stimulus. In different blocks of trials, standard stimuli were either 600 or 2000 ms. Behavioral measures included the point of subjective equality, a measure of accuracy, and the coefficient of variation, a measure of variability. Lesions of the right middle and inferior frontal gyri were associated with decrements in performance on both durations. In addition, lesions of the left temporal lobe and right precentral gyrus were associated exclusively with impaired performance for subsecond stimuli. In line with results from other studies, these data suggest that different circuits are necessary for timing intervals in these ranges, and that right frontal areas are particularly important to timing.

Oxycodone lengthens reproductions of suprasecond time intervals in human research volunteers.

Oxycodone, a popularly used opioid for treating pain, is widely abused. Other drugs of abuse have been shown to affect time perception, which, in turn, may affect sensitivity to future consequences. This may contribute to continued use. This study evaluated the effect of oxycodone on time perception in normal healthy volunteers. For this within-subject, double-blind design study, participants performed a temporal reproduction task before and after receiving placebo or oxycodone (15 mg, orally) over six outpatient sessions. Participants were first trained with feedback to reproduce three standard intervals (1.1, 2.2, and 3.3 s) in separate blocks by matching response latency from a start signal to the duration of that block's standard interval. During testing, participants were instructed to reproduce the three intervals from memory without feedback before and after drug administration. Oxycodone significantly lengthened time estimations for the two longer intervals relative to placebo. These results suggest that opioids alter temporal processing for intervals greater than 1 s, raising questions about the effect of these drugs on the valuation of future consequences.

The use of mesenchymal stem cells in collagen-based scaffolds for tissue-engineered repair of tendons.

Tendon and ligament injuries are significant contributors to musculoskeletal injuries. Unfortunately, traditional methods of repair are not uniformly successful and can require revision surgery. Our research is focused on identifying appropriate animal injury models and using tissue-engineered constructs (TECs) from bone-marrow-derived mesenchymal stem cells and collagen scaffolds. Critical to this effort has been the development of functional tissue engineering (FTE). We first determine the in vivo mechanical environment acting on the tissue and then precondition the TECs in culture with aspects of these mechanical signals to improve repair outcome significantly. We describe here a detailed protocol for conducting several complete iterations around our FTE 'road map.' The in vitro portion, from bone marrow harvest to TEC collection, takes 54 d. The in vivo portion, from TEC implantation to limb harvest, takes 84 d. One complete loop around the tissue engineering road map, as presented here, takes 138 d to complete.

Interval timing disruptions in subjects with cerebellar lesions.

The cerebellum has long been implicated in time perception, particularly in the subsecond range. The current set of studies examines the role of the cerebellum in suprasecond timing, using analysis of behavioral data in subjects with cerebellar lesions. Eleven cerebellar lesion subjects and 17 controls were tested on temporal estimation, reproduction and production, for times ranging from 2 to 12s. Cerebellar patients overproduced times on both the reproduction and production tasks; the effect was greatest at the shortest duration. A subset of patients also underestimated intervals. Cerebellar patients were significantly more variable on the estimation and reproduction tasks. No significant differences between normal and cerebellar patients were found on temporal discrimination tasks with either sub- or suprasecond times. Patients with damage to the lateral superior hemispheres or the dentate nuclei showed more significant impairments than those with damage elsewhere in the cerebellum, and patients with damage to the left cerebellum had more significant differences from controls than those with damage to the right. These data suggest that damage to the middle-to-superior lobules or the left hemisphere is especially detrimental to timing suprasecond intervals. We suggest that this region be considered part of a network of brain structures including the DLPFC that is crucial for interval timing.

Averaging of temporal memories by rats.

Rats were trained on a mixed fixed-interval schedule in which stimulus A (tone or light) indicated food availability after 10 s and stimulus B (the other stimulus) indicated food availability after 20 s. Testing consisted of nonreinforced probe trials in which the stimulus was A, B, or the compound AB. On single-stimulus trials, rats responded with a peak of activity around the programmed reinforced time. On compound-stimulus trials, rats showed a single scalar peak of responding at a time midway between those for stimulus A and B. These results suggest that when provided with discrepant information regarding the temporal predictability of reinforcement, rats compute an average of the scheduled reinforcement times for the A and B stimuli and use this average to generate an expectation of reward for the compound stimuli.

Three-dimensional in vitro effects of compression and time in culture on aggregate modulus and on gene expression and protein content of collagen type II in murine chondrocytes.

The objectives of this study were to determine how culture time and dynamic compression, applied to murine chondrocyte-agarose constructs, influence construct stiffness, expression of col2 and type II collagen. Chondrocytes were harvested from the ribs of six newborn double transgenic mice carrying transgenes that use enhanced cyan fluorescent protein (ECFP) and green fluorescent protein (GFP-T) as reporters for expression from the col2a1 and col1a1 promoters, respectively. Sixty-three constructs (8 mm diameter x 3 mm thick) per animal were created by seeding chondrocytes (10 x 10(6) per mL) in agarose gel (2% w/v). Twenty-eight constructs from each animal were stimulated for 7, 14, 21, or 28 days in a custom bioreactor housed in an electromagnetic system. Twenty-eight constructs exposed to identical culture conditions but without mechanical stimulation served as nonstimulated controls for 7, 14, 21, and 28 days. The remaining seven constructs served as day 0 controls. Fluorescing cells with rounded morphology were present in all constructs at all five time points. Seven, 14, 21, and 28 days of stimulation significantly increased col2 expression according to ECFP fluorescence and messenger RNA expression according to quantitative reverse transcriptase polymerase chain reaction. Col2 gene expression in stimulated and nonstimulated constructs showed initial increases up to day 14 and then showed decreases by day 28. Stimulation significantly increased type II collagen content at 21 and 28 days and aggregate modulus only at 28 days. There was a significant increase in aggregate modulus in stimulated constructs between day 0 and 7 and between day 21 and day 28. This study reveals that compressive mechanical stimulation is a potent stimulator of col2 gene expression that leads to measurable but delayed increases in protein (type II collagen) and then biomechanical stiffness. Future studies will examine the effects of components of the mechanical signal in culture and address the question of whether such in vitro improvements in tissue-engineered constructs enhance repair outcomes after surgery.

Combined effects of scaffold stiffening and mechanical preconditioning cycles on construct biomechanics, gene expression, and tendon repair biomechanics.

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits of stimulation among repairs postsurgery. Our findings emphasize the importance of properly preconditioning a scaffold to better control/modulate MSC differentiation in vitro and to further enhance repair outcome in vivo.

Tensile stimulation of murine stem cell-collagen sponge constructs increases collagen type I gene expression and linear stiffness.

The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h=day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.

Evidence for age-related changes to temporal attention and memory from the choice time production task.

The effect of aging on interval timing was examined using a choice time production task, which required participants to choose a key response based on the location of the stimulus, but to delay responding until after a learned time interval. Experiment 1 varied attentional demands of the response choice portion of the task by varying difficulty of stimulus-response mapping. Choice difficulty affected temporal accuracy equally in both age groups, but older participants' response latencies were more variable under more difficult response choice conditions. Experiment 2 tested the contribution of long-term memory to differences in choice time production between age groups over 3 days of testing. Direction of errors in time production between the two age groups diverged over the 3 sessions, but variability did not differ. Results from each experiment separately show age-related changes to attention and memory in temporal processing using different measures and manipulations in the same task.

Improving linear stiffness of the cell-seeded collagen sponge constructs by varying the components of the mechanical stimulus.

In vitro mechanical stimulation has been reported to induce cell alignment and increase cellular proliferation and collagen synthesis. Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases construct stiffness and repair biomechanics after surgery. However, these studies used a single mechanical stimulation profile, the latter composed of multiple components whose individual and combined effects on construct properties remain unknown. Thus, the purpose of this study was to understand the relative importance of a subset of these components on construct stiffness. To try to optimize the resulting mechanical stimulus, we used an iterative process to vary peak strain, cycle number, and cycle repetition while controlling cycle frequency (1 Hz), rise and fall times (25% and 17% of the period, respectively), hours of stimulation/day (8 h/day), and total time of stimulation (12 days). Two levels of peak strain (1.2 % and 2.4%), cycle number (100 and 3000 cycles/day), and cycle repetition (1 and 20) were first examined. Higher levels of peak strain and cycle number were then examined to optimize the stimulus using response surface methodology. Our results indicate that constructs stimulated with 2.4% strain, 3000 cycles/day, and one cycle repetition produced the stiffest constructs. Given the significant positive correlations we have previously found between construct stiffness and repair biomechanics at 12 weeks post-surgery, these in vitro enhancements offer the prospect of further improving repair biomechanics.

Functional tissue engineering for tendon repair: A multidisciplinary strategy using mesenchymal stem cells, bioscaffolds, and mechanical stimulation.

Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell-gel-suture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end-posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load-bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury.

Mechanical stimulation of tendon tissue engineered constructs: effects on construct stiffness, repair biomechanics, and their correlation.

The objective of this study was to determine how in vitro mechanical stimulation of tissue engineered constructs affects their stiffness and modulus in culture and tendon repair biomechanics 12 weeks after surgical implantation. Using six female adult New Zealand White rabbits, autogenous tissue engineered constructs were created by seeding mesenchymal stem cells (0.1 x 10(6) cells/ml) in collagen gel (2.6 mg/ml) and combining both with a collagen sponge. Employing a novel experimental design strategy, four constructs from each animal were mechanically stimulated (one 1 Hz cycle every 5 min to 2.4% peak strain for 8 h/day for 2 weeks) while the other four remained unstretched during the 2 week culture period. At the end of incubation, three of the mechanically stimulated (S) and three of the nonstimulated (NS) constructs from each animal were assigned for in vitro mechanical testing while the other two autogenous constructs were implanted into bilateral full-thickness, full-length defects created in the central third of rabbit patellar tendons (PTs). No significant differences were found in the in vitro linear stiffnesses between the S (0.15+/-0.1 N/mm) and NS constructs (0.08+/-0.02 N/mm; mean+/-SD). However, in vitro mechanical stimulation significantly increased the structural and material properties of the repair tissue, including a 14% increase in maximum force (p=0.01), a 50% increase in linear stiffness (p=0.001), and 23-41% increases in maximum stress and modulus (p=0.01). The S repairs achieved 65%, 80%, 60%, and 40% of normal central PT maximum force, linear stiffness, maximum stress, and linear modulus, respectively. The results for the S constructs exceed values obtained previously by our group using the same animal and defect model, and to our knowledge, this is the first study to show the benefits of in vitro mechanical stimulation on tendon repair biomechanics. In addition, the linear stiffnesses for the construct and repair were positively correlated (r=0.56) as were their linear moduli (r=0.68). Such in vitro predictors of in vivo outcome hold the potential to speed the development of tissue engineered products by reducing the time and costs of in vivo studies.