Three-Dimensional Modeling of the Structural Microenvironment in Post-Traumatic War Wounds.

BACKGROUND: The development of post-traumatic heterotopic ossification (HO) is a common, undesirable sequela in patients with high-energy (war-related) extremity injuries. While inflammatory and osteoinductive signaling pathways are known to be involved in the development and progression of post-traumatic HO, features of the structural microenvironment within which the ectopic bone begins to form remain poorly understood. Thus, increasing our knowledge of molecular and structural changes within the healing wound may help elucidate the pathogenesis of post-traumatic HO and aid in the development of specific treatment and/or prevention strategies. METHODS: In this study, we performed high-resolution microscopy and biochemical analysis of tissues obtained from traumatic war wounds to characterize changes in the structural microenvironment. In addition, using an electrospinning approach, we modeled this microenvironment to reconstitute a three-dimensional type I collagen scaffold with non-woven, randomly oriented nanofibers where we evaluated the performance of primary mesenchymal progenitor cells. RESULTS: We found that traumatic war wounds are characterized by a disorganized, densely fibrotic collagen I matrix that influences progenitor cells adhesion, proliferation and osteogenic differentiation potential. CONCLUSION: Altogether, these results suggest that the structural microenvironment present in traumatic war wounds has the potential to contribute to the development of post-traumatic HO. Our findings may support novel treatment strategies directed towards modifying the structural microenvironment after traumatic injury.

In vivo model of human post-traumatic heterotopic ossification demonstrates early fibroproliferative signature.

BACKGROUND: The relationship between the tissue injury healing response and development of heterotopic ossification (HO) is poorly understood. Here we compare a rat blast model and human traumatized muscle from a blast injury to study the early signatures of osteogenesis and fibrosis during the formation of HO. METHODS: Rat and human tissues were characterized using histology, scanning electron microscopy, immunohistochemistry, as well as gene and protein expression analysis. Additionally, animals and humans were assessed radiographically for HO formation following injury. RESULTS: Markers of bone formation were dramatically increased in tissue samples from both humans and rats, and both displayed increased fibroproliferative regions within the injured tissues and elevated expression of markers of tissue fibrosis such as TGF-ß1, Fibronectin, SMAD3 and PAI-1. Markers of inflammation and fibrosis (ACTA, TNFα, BMP1 and BMP3) were elevated at the RNA level in both rat and human samples. By day 42, bone formation in the rat blast model appeared similar in radiographs compared to human patients who progressed to develop post-traumatic HO. CONCLUSIONS: Our data demonstrates that a similar early fibrotic response is evident in both the rat blast model and the human tissues following a traumatic injury and demonstrates the relevance of this animal model for future translational studies.

Characterization of discrete subpopulations of progenitor cells in traumatic human extremity wounds.

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.

Stem cell applications in military medicine.

There are many similarities between health issues affecting military and civilian patient populations, with the exception of the relatively small but vital segment of active soldiers who experience high-energy blast injuries during combat. A rising incidence of major injuries from explosive devices in recent campaigns has further complicated treatment and recovery, highlighting the need for tissue regenerative options and intensifying interest in the possible role of stem cells for military medicine. In this review we outline the array of tissue-specific injuries typically seen in modern combat - as well as address a few complications unique to soldiers--and discuss the state of current stem cell research in addressing each area. Embryonic, induced-pluripotent and adult stem cell sources are defined, along with advantages and disadvantages unique to each cell type. More detailed stem cell sources are described in the context of each tissue of interest, including neural, cardiopulmonary, musculoskeletal and sensory tissues, with brief discussion of their potential role in regenerative medicine moving forward. Additional commentary is given to military stem cell applications aside from regenerative medicine, such as blood pharming, immunomodulation and drug screening, with an overview of stem cell banking and the unique opportunity provided by the military and civilian overlap of stem cell research.

Heterotopic ossification following musculoskeletal trauma: modeling stem and progenitor cells in their microenvironment.

Heterotopic ossification (HO), characterized by the formation of mature bone in the soft tissues, is a complication that can accompany musculoskeletal injury, and it is a frequent occurrence within the military population that has experienced orthopaedic combat trauma. The etiology of this disease is largely unknown. Our laboratory has developed strategies to investigate the cellular and molecular events leading to HO using clinical specimens that were obtained during irrigation and debridement of musculoskeletal injuries. Our approach enables to study (1) the cell types that are responsible for pathological transformation and ossification, (2) the cell- and tissue-level signaling that induces the pathologic transformation, and (3) the effect of extracellular matrix topography and force transduction on HO progression. In this review, we will report on our findings in each of these aspects of HO etiology and describe our efforts to recapitulate our findings in an animal model for traumatic HO.

Nanofiber matrices promote the neuronal differentiation of human embryonic stem cell-derived neural precursors in vitro.

The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

The effect of nanofibre surface amine density and conjugate structure on the adhesion and proliferation of human haematopoietic progenitor cells.

Previous studies have shown that substrate surface chemistry and topography exhibit significant impact on haematopoietic progenitor cell adhesion, proliferation and differentiation. In the present study, the effect of surface amine density and structure of grafted polymer chains on the adhesion and expansion of haematopoietic progenitor cells was investigated. Cryopreserved human umbilical cord blood CD133(+) cells were expanded in cytokine-supplemented medium on ethylenediamine (EDA)- or 2-aminoethyl methacrylate hydrochloride (AEMA)-grafted polyethersulphone (PES) nanofibre scaffolds for 10 days. Although the percentage of CD34(+) cells among the expanded cells increased with the surface amine density, the maximum fold expansion of CD34(+) cells was obtained at a moderate amine density of 20-80 nmol cm(-2). When comparing nanofibre matrices with similar amine densities, but prepared with two different methods, cells cultured on the AEMA-grafted PES nanofibre matrix showed lower fold expansion in terms of total cell number (300 ± 84 fold) and CD34(+) cell number (68 ± 19-fold) in comparison with those cultured on EDA-grafted nanofibres (787 ± 84-fold and 185 ± 84-fold, respectively). These results indicate that the surface amine density and the conjugate structure are important determinants for the preservation of CD34 surface marker and expansion efficiency of CD34(+) cells.

The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation.

Neural stem/progenitor cells (NSCs) are capable of self-renewal and differentiation into all types of neural lineage under different biochemical and topographical cues. In this study, we cultured rat hippocampus-derived adult NSCs (rNSCs) on laminin-coated electrospun Polyethersulfone (PES) fiber meshes with average fiber diameters of 283+/-45 nm, 749+/-153 nm and 1452+/-312 nm; and demonstrated that fiber diameter of PES mesh significantly influences rNSC differentiation and proliferation. Under the differentiation condition (in the presence of 1 microM retinoic acid and 1% fetal bovine serum), rNSCs showed a 40% increase in oligodendrocyte differentiation on 283-nm fibers and 20% increase in neuronal differentiation on 749-nm fibers, in comparison to tissue culture polystyrene surface. SEM imaging revealed that cells stretched multi-directionally to follow underlying 283-nm fibers, but extended along a single fiber axis on larger fibers. When cultured on fiber meshes in serum free medium in the presence of 20 ng/mL of FGF-2, rNSCs showed lower proliferation and more rounded morphology compared to that cultured on laminin-coated 2D surface. As the fiber diameter decreased, higher degree of proliferation and cell spreading and lower degree of cell aggregation were observed. This collective evidence indicates fiber topography can play a vital role in regulating differentiation and proliferation of rNSCs in culture.

Effect of microcomputed tomography voxel size on the finite element model accuracy for human cancellous bone.

The level of structural detail that can be acquired and incorporated in a finite element (FE) analysis might greatly influence the results of microcomputed tomography (microCT)-based FE simulations, especially when relatively large bones, such as whole vertebrae, are of concern. We evaluated the effect of scanning and reconstruction voxel size on the microCT-based FE analyses of human cancellous tissue samples for fixed- and free-end boundary conditions using different combinations of scan/reconstruction voxel size. We found that the bone volume fraction (BV/TV) did not differ considerably between images scanned at 21 and 50 microm and reconstructed at 21, 50, or 110 microm (-0.5% to 7.8% change from the 21/21 microm case). For the images scanned and reconstructed at 110 microm, however, there was a large increase in BV/TV compared to the 21/21 microm case (58.7%). Fixed-end boundary conditions resulted in 1.8% [coefficient of variation (COV)] to 14.6% (E) difference from the free-end case. Dependence of model output parameters on scanning and reconstruction voxel size was similar between free- and fixed-end simulations. Up to 26%, 30%, 17.8%, and 32.3% difference in modulus (E), and average (VMExp), standard deviation (VMSD) and coefficient of variation (COV) of von Mises stresses, respectively, was observed between the 21/21 microm case and other scan/reconstruction combinations within the same (free or fixed) simulation group. Observed differences were largely attributable to scanning resolution, although reconstruction resolution also contributed significantly at the largest voxel sizes. All 21/21 microm results (taken as the gold standard) could be predicted from the 21/50 (r2adj= 0.91-0.99;p<0.001), 21/110 (r2adj =0.58-0.99;p<0.02) and 50/50 results (r2adj=0.61-0.97;p<0.02). While BV/TV, VMSD, and VMExp/sigma(z) from the 21/21 could be predicted by those from the 50/110 (r2adj =0.63-0.93;p<0.02) and 110/110 (r2adj =0.41-0.77;p<0.05) simulations as well, prediction of E, VMExp, and COV became marginally significant (0.04

The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone.

Stereological parameters have been used as an approximation for the architecture of trabecular bone. Structural indices such as bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), bone surface-to-volume ratio (BS/BV), degree of anisotropy (MIL1/MIL3), and connectivity density (-Euler/Vol) have been widely studied to investigate pathological conditions in bone. Due to its high resolution and nondestructiveness, microcomputed tomography (micro-CT) has been utilized to take precise three-dimensional (3D) images of trabecular microstructures. However, spatial limitations for applying micro-CT-based analyses to large specimens, such as whole vertebral bodies, require using larger scanning and reconstruction voxel sizes. In this study, combinations of three different scanning and reconstruction voxel size were used to represent best possible voxel size (21 microm; best in our scanner for the specimen size used) relative to other voxel sizes used in this study, commonly used intermediate voxel sizes (50 microm), and those applicable to scans of whole human vertebral bodies (110 microm) in order to examine the effect of scanning and reconstruction voxel size on stereological measures for human cancellous bone. The error in stereological parameters calculated using combinations of large voxel sizes compared to the gold standard (best possible case) ranged from 0.1% to 102%. The signed magnitude of the error in other cases relative to the gold standard was a function of either scanning or reconstruction voxel size or both (r2=0.55-0.95). For most of the structural indices, the results from analysis of images with larger voxel sizes were correlated with those from the gold standard (r2=0.55-0.99) except for Tb.N at 110/110 microm, MIL1/MIL3 at larger than 110 microm reconstruction voxel size, and -Euler/Vol at any combination of voxel sizes. Overall, it was observed that resampling a high resolution image at lower resolutions (corresponding to increasing reconstruction voxel size in this study) had different effects on the calculated parameters than scanning at the same low resolution (corresponding to increasing scanning voxel size in this study). Our results show that investigations of image resolution should include actual scans at the resolution of interest rather than simply coarsening of high-resolution images as is customarily done.

Apparent viscoelastic anisotropy as measured from nondestructive oscillatory tests can reflect the presence of a flaw in cortical bone.

There is evidence that damage, viscoelastic stiffness properties, and postyield mechanical properties are related in bone tissue. Our objective was to test whether presence of a flaw would have an influence on the apparent viscoelastic properties of bone. Examining the effect of flaw orientation on apparent viscoelastic properties and utilization of dynamic mechanical analysis (DMA) as a nondestructive means for detection of damage were our secondary objectives. Cortical bone beams (2 x 2 x 19 mm) machined from the cranial cortex of the radii of six Warhill sheep were used. The specimens were placed in a DMA machine and baseline measurements of storage modulus (E1) and loss factor (tan delta), once for loads in the craniocaudal and once in the mediolateral directions, were performed using a three-point bending configuration for a frequency range of 1-10 Hz. Craniocaudal/mediolateral measurement ratio was calculated as a measure of anisotropy for tan delta and E1. After cutting a thin through-thickness macroscopic notch on the caudal surface at the center of each beam, oscillatory tests were repeated. Two-way repeated measures analysis of variance followed by Tukey's test was used with group (craniocaudal, mediolateral, notched craniocaudal, and notched mediolateral measurements) and frequency as factors. Regression analysis and analysis of covariance were used for examining the relationship between viscoelastic parameters and frequency. Tan delta and E1 were not different between craniocaudal and mediolateral measurements before the flaw was introduced (p > 0.8 and p = 1, respectively). In the presence of the flaw, tan delta was significantly increased (p < 0.003) whereas E1 was significantly reduced (p < 0.001) for craniocaudal measurements. Tan delta and E1 were nearly isotropic in the tested directions before the introduction of a flaw into the bone tissue. Introduction of a flaw resulted in increased tan delta and E1 anisotropy. Presence of a notch resulted in a significant increase in tan delta anisotropy with increasing frequency. In conclusion, we have demonstrated that cortical bone tissue exhibits a different apparent viscoelastic behavior in the presence of a flaw and depending on the flaw's orientation. Our finding that the presence of a notch and its orientation can be detected by nondestructive DMA suggests that in vivo techniques may be developed for detection of cortical bone damage.