A finite element inverse analysis to assess functional improvement during the fracture healing process

Assessment of the restoration of load-bearing function is the central goal in the study of fracture healing process. During the fracture healing, two critical aspects affect its analysis: (1) material properties of the callus components, and (2) the spatio-temporal architecture of the callus with respect to cartilage and new bone formation. In this study, an inverse problem methodology is used which takes into account both features and yields material property estimates that can analyze the healing changes. Six stabilized fractured mouse tibias are obtained at two time points during the most active phase of the healing process, respectively 10 days (n=3), and 14 days (n=3) after fracture. Under the same displacement conditions, the inverse procedure estimations of the callus material properties are generated and compared to other fracture healing metrics. The FEA estimated property is the only metric shown to be statistically significant (p=0.0194) in detecting the changes in the stiffness that occur during the healing time points. In addition, simulation studies regarding sensitivity to initial guess and noise are presented; as well as the influence of callus architecture on the FEA estimated material property metric. The finite element model inverse analysis developed can be used to determine the effects of genetics or therapeutic manipulations on fracture healing in rodents

Leveraging Mathematical Modeling to Quantify Pharmacokinetic and Pharmacodynamic Pathways: Equivalent Dose Metric

Treatment response assays are often summarized by sigmoidal functions comparing cell survival at a single timepoint to applied drug concentration. This approach has a limited biophysical basis, thereby reducing the biological insight gained from such analysis. In particular, drug pharmacokinetic and pharmacodynamic (PK/PD) properties are overlooked in developing treatment response assays, and the accompanying summary statistics conflate these processes. Here, we utilize mathematical modeling to decouple and quantify PK/PD pathways. We experimentally modulate specific pathways with small molecule inhibitors and filter the results with mechanistic mathematical models to obtain quantitative measures of those pathways. Specifically, we investigate the response of cells to time-varying doxorubicin treatments, modulating doxorubicin pharmacology with small molecules that inhibit doxorubicin efflux from cells and DNA repair pathways. We highlight the practical utility of this approach through proposal of the “equivalent dose metric.” This metric, derived from a mechanistic PK/PD model, provides a biophysically-based measure of drug effect. We define equivalent dose as the functional concentration of drug that is bound to the nucleus following therapy. This metric can be used to quantify drivers of treatment response and potentially guide dosing of combination therapies. We leverage the equivalent dose metric to quantify the specific intracellular effects of these small molecule inhibitors using population-scale measurements, and to compare treatment response in cell lines differing in expression of drug efflux pumps. More generally, this approach can be leveraged to quantify the effects of various pharmaceutical and biologic perturbations on treatment response

Comparison of microCT and an inverse finite element approach for biomechanical analysis: Results in a MSC therapeutic system for fracture healing

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures

Mesenchymal Stem Cells Expressing Insulin-like Growth Factor-I (MSCIGF) Promote Fracture Healing and Restore New Bone Formation in Irs1 Knockout Mice: Analyses of MSCIGF Autocrine and Paracrine Regenerative Effects

Failures of fracture repair (non-unions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe and feasible. The contributions of MSC to the reparative process can occur through autocrine as well as paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSC expressing insulin-like growth factor-I (MSCIGF), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and non-unions. MSCIGF or traceable MSCIGF-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knock-out (Irs1−/−) mice with a stabilized tibia fracture. Healing was assed using biomechanical testing, micro-computed-tomography (µCT) and histological analyses. We found that systemically transplanted MSCIGF through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSCIGF to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced induced osteoglastogenesis in MSC was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1−/− mice as a non-union fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap; a callus that basically absent in Irs1−/− left untransplanted or transplanted with MSC. We provided evidence of effects and mechanisms for transplanted MSCIGF in fracture repair and potentially to treat non-unions

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I + MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I + MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I + MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I + MSC could be a novel therapeutic approach in patients who have inadequate fracture repair

Regenerative Effects of Transplanted Mesenchymal Stem Cells in Fracture Healing

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat non-unions. The use of MSC to treat fractures is attractive as it would be implementing a reparative process that should be in place but occurs to be defective or protracted and MSC effects would be needed only for the repairing time that is relatively brief. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed-tomography and histological studies. Transplanting CMV-Cre-R26R-LacZ-MSC, we found that MSC engrafted within the callus endosteal niche. Using MSC from BMP-2-Lac-Z mice genetically modified using a bacterial artificial chromosome system to be β-gal reporters for BMP-2 expression, we found that MSC contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow to design novel MSC-based therapies to treat fractures

Impaired DNA damage response signaling by FUS-NLS mutations leads to neurodegeneration and FUS aggregate formation

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS

Quantifying Mechanical Properties in a Murine Fracture Healing System Using Inverse Modeling: Preliminary Work

ABSTRACT Understanding bone remodeling and mechanical property characteristics is important for assessing treatments to accelerate healing or in developing diagnostics to evaluate successful return to function. The murine system whereby mid-diaphaseal tibia fractures are imparted on the subject and fracture healing is assessed at different time points and under different therapeutic conditions is a particularly useful model to study. In this work, a novel inverse geometric nonlinear elasticity modeling framework is proposed that can reconstruct multiple mechanical properties from uniaxial testing data. To test this framework, the Lame' constants were reconstructed within the context of a murine cohort (n=6) where there were no differences in treatment post tibia fracture except that half of the mice were allowed to heal 4 days longer (10 day, and 14 day healing time point, respectively). The properties reconstructed were a shear modulus of G=511.2 ± 295.6 kPa, and 833.3± 352.3 kPa for the 10 day, and 14 day time points respectively. The second Lame' constant reconstructed at =1002.9 ±42.9 kPa, and 14893.7 ± 863.3 kPa for the 10 day, and 14 day time points respectively. An unpaired Student t-test was used to test for statistically significant differences among the groups. While the shear modulus did not meet our criteria for significance, the second Lame' constant did at a value p<0.0001. Traditional metrics that are commonly used within the bone fracture healing research community were not found to be statistically significant

Precision Medicine with Imprecise Therapy: Computational Modeling for Chemotherapy in Breast Cancer

Medical oncology is in need of a mathematical modeling toolkit that can leverage clinically-available measurements to optimize treatment selection and schedules for patients. Just as the therapeutic choice has been optimized to match tumor genetics, the delivery of those therapeutics should be optimized based on patient-specific pharmacokinetic/pharmacodynamic properties. Under the current approach to treatment response planning and assessment, there does not exist an efficient method to consolidate biomarker changes into a holistic understanding of treatment response. While the majority of research on chemotherapies focus on cellular and genetic mechanisms of resistance, there are numerous patient-specific and tumor-specific measures that contribute to treatment response. New approaches that consolidate multimodal information into actionable data are needed. Mathematical modeling offers a solution to this problem. In this perspective, we first focus on the particular case of breast cancer to highlight how mathematical models have shaped the current approaches to treatment. Then we compare chemotherapy to radiation therapy. Finally, we identify opportunities to improve chemotherapy treatments using the model of radiation therapy. We posit that mathematical models can improve the application of anticancer therapeutics in the era of precision medicine. By highlighting a number of historical examples of the contributions of mathematical models to cancer therapy, we hope that this contribution serves to engage investigators who may not have previously considered how mathematical modeling can provide real insights into breast cancer therapy

Heterogeneity in mouse spasmolytic polypeptide-expressing metaplasia lineages identifies markers of metaplastic progression

Objectives: Spasmolytic polypeptide-expressing metaplasia (SPEM) develops as a preneoplastic lesion in the stomachs of mice and humans after parietal cell loss. To identify the commonalities and differences between phenotypic SPEM lineages, SPEM were studied from three different mouse models of parietal cell loss: with chronic inflammation with Helicobacter felis infection; with acute inflammation with L635 treatment; and without inflammation following DMP-777 treatment. Design: RNA transcripts from laser capture microdissected normal chief cells and SPEM lineages were compared using gene microarray. Alterations in transcripts were validated by quantitative real-time PCR. Clusterin and cystic fibrosis transmembrane conductance regulator (CFTR) were selected for immunohistochemical analysis in all mouse models as well as in human SPEM, intestinal metaplasia and gastric cancer. Results: Transcript expression patterns demonstrated differences among the phenotypic SPEM models. Clusterin expression was significantly upregulated in all three mouse SPEM models as well as in human SPEM. The highest clusterin expression in human gastric cancers correlated with poor survival. Conversely, CFTR expression was upregulated only in SPEM with inflammation in mice. In humans, intestinal metaplasia, but not SPEM, expressed CFTR. Conclusions: While markers such as clusterin are expressed in all phenotypic SPEM lineages, distinct patterns of upregulated genes including CFTR are present in murine metaplasia associated with inflammation, indicative of progression of metaplasia towards a more intestinalised metaplastic phenotype.National Institutes of Health (U.S.) (grant RO1 DK071590)United States. American Recovery and Reinvestment Act of 2009 (ARRA supplement (DK071590-S1))National Institutes of Health (U.S.) (NIH grant RO1 AI037750)National Institutes of Health (U.S.) (NIH grant P30 ES02109)National Institutes of Health (U.S.) (NIH grant R01 DK 077065)National Institutes of Health (U.S.) (NIH grant P50 CA95060)Vanderbilt Digestive Disease Center (P30 DK058404)Vanderbilt-Ingram Cancer Cente