Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation.

Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.

Akt-mediated mechanotransduction in murine fibroblasts during hypertrophic scar formation.

Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt-mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide-3-kinase/Akt inhibitor). We also demonstrated that high-frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α-smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.

IFATS collection: Adipose stromal cells adopt a proangiogenic phenotype under the influence of hypoxia.

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.

Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis.

Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism.

Biomechanical comparison: stability of lateral-approach anterior lumbar interbody fusion and lateral fixation compared with anterior-approach anterior lumbar interbody fusion and posterior fixation in the lower lumbar spine.

OBJECT: The stability of lateral lumbar interbody graft-augmented fusion and supplementary lateral plate fixation in human cadavers has not been determined. The purpose of this study was to investigate the immediate biomechanical stabilities of the following: 1) femoral ring allograft (FRA)-augmented anterior lumbar interbody fusion (ALIF) after left lateral discectomy combined with additional lateral MACS HMA plate and screw fixation; and 2) ALIF combined with posterior transpedicular fixation after anterior discectomy. METHODS: Sixteen human lumbosacral spines were loaded with six modes of motion. The intervertebral motion was measured using a video-based motion-capturing system. The range of motion (ROM) and the neutral zone (NZ) in each loading mode were compared with a maximum of 7.5 Nm. The ROM values for both stand-alone ALIF approaches were similar to those of the intact spine, whereas NZ measurements were higher in most loading modes. No significant intergroup differences were found. The ROM and NZ values for lateral fixation in all modes were significantly lower than those of intact spine, except when NZ was measured in lateral bending. All ROM and NZ values for transpedicular fixation were significantly lower than those for stand-alone anterior ALIF. Transpedicular fixation conferred better stabilization than lateral fixation in flexion, extension, and lateral bending modes. CONCLUSIONS: Neither approach to stand-alone FRA-augmented ALIF provided sufficient stabilization, but supplementary instrumentation conferred significant stabilization. The MACS HMA plate and screw fixation system, although inferior to posterior transpedicular fixation, provided adequate stability compared with the intact spine and can serve as a sound alternative to supplementary spinal stabilization.

Exercise induces stromal cell-derived factor-1α-mediated release of endothelial progenitor cells with increased vasculogenic function.

BACKGROUND: Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline. METHODS: Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated. RESULTS: Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation. CONCLUSIONS: Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.

Biomechanical comparison of anterior and posterior stabilization methods in atlantoaxial instability.

OBJECT: The authors compared the biomechanical stability of two anterior fixation procedures--anterior C1-2 Harms plate/screw (AHPS) fixation and the anterior C1-2 transarticular screw (ATS) fixation; and two posterior fixation procedures--the posterior C-1 lateral mass combined with C-2 pedicle screw/rod (PLM/APSR) fixation and the posterior C1-2 transarticular screw (PTS) fixation after destabilization. METHODS: Sixteen human cervical spine specimens (Oc-C3) were tested in three-dimensional flexion-extension, axial rotation, and lateral bending motions after destabilization by using an atlantoaxial C1-2 instability model. In each loading mode, moments were applied to a maximum of 1.5 Nm, and the range of motion (ROM), neutral zone (NZ), and elastic zone (EZ) were determined and values compared using the intact spine, the destabilized spine, and the postfixation spine. The AHPS method produced inferior biomechanical results in flexion-extension and lateral bending modes compared with the intact spine. The lateral bending NZ and ROM for this method differed significantly from the other three fixation techniques (p < 0.05), although statistically significant differences were not obtained for all other values of ROM and NZ for the other three procedures. The remaining three methods restored biomechanical stability and improved it over that of the intact spine. CONCLUSIONS: The PLM/APSR fixation method was found to have the highest biomechanical stiffness followed by PTS, ATS, and AHPS fixation. The PLM/APSR fixation and AATS methods can be considered good procedures for stabilizing the atlantoaxial joints, although specific fixation methods are determined by the proper clinical and radiological characteristics in each patient.

A biomechanical comparison of supplementary posterior translaminar facet and transfacetopedicular screw fixation after anterior lumbar interbody fusion.

OBJECT: Facet screw fixation is the lowest profile lumbar stabilization method. In this study the immediate biomechanical stability provided by the two different types of fixation are compared: translaminar facet screw (TLFS) and transfacetopedicular screw (TFPS) placement after anterior lumbar interbody fusion (ALIF) using a femoral ring allograft. Both facet screw fixation types were also compared with the gold standard, transpedicular screw and rod (TSR) fixation. METHODS: Twenty-four human lumbosacral spines were tested in the following sequence: intact state, after discectomy, after ALIF, and after TLFS, TFPS, or TSR fixation. Intervertebral motions were measured by a video-based motion capture system. The range of motion (ROM) and neutral zone (NZ) were compared for each loading to a maximum of 7.5 Nm. The ROMs for stand-alone ALIFs were less than but similar to those of the intact spine, but NZs were slightly increased in all modes. The ROMs for both TLFS and TFPS fixation were significantly decreased from those of the intact spine in all modes and those of the stand-alone ALIF in flexion and extension. The TLFS and TFPS fixations significantly reduced NZs to below that of the intact spine in all modes. Compared with NZs for ALIF, both types of fixation revealed significantly lower values, except for TLFS placement in lateral bending and TFPS fixation in lateral bending and rotation. There were no significant differences between TLFS and TFPS fixation. There were also no significant differences among both TLFS and TFPS and TSR fixations, except that TFPS was inferior to TSR in lateral bending. CONCLUSIONS: Stand-alone ALIF may not provide sufficient stability. Both facet fixations produced significant additional stability and both are comparable to TSR fixation. Although TFPS fixation revealed a slightly inferior result, TFPSs can be placed percutaneously with the assistance of fluoroscopic guidance and it makes the posterior facet fixation minimally invasive. Therefore, the TFPS fixation can be considered as a good alternative to TLFS fixation.

A biomechanical comparison of three surgical approaches in bilateral subaxial cervical facet dislocation.

OBJECT: In bilateral cervical facet dislocation, biomechanical stabilities between anterior locking screw/plate fixation after anterior cervical discectomy and fusion (ACDFP) and posterior transpedicular screw/rod fixation after anterior cervical discectomy and fusion (ACDFTP) have not been compared using the human cadaver, although ACDFP has been performed frequently. In this study the stability of ACDFP, a posterior wiring procedure after ACDFP (ACDFPW), and ACDFTP for treatment of bilateral cervical facet dislocation were compared. METHODS: Spines (C3-T1) from 10 human cadavers were tested in the intact state, and then after ACDFP, ACDFPW, and ACDFTP were performed. Intervertebral motion was measured using a video-based motion capture system. The range of motion (ROM) and neutral zone (NZ) were compared for each loading mode to a maximum of 2 Nm. The ROM for spines treated with ACDFP was below that of the intact spine in all loading modes, with statistical significance in flexion and extension, but NZs were decreased in flexion and extension and slightly increased in bending and axial rotation; none of these showed statistical significance. The ACDFPW produced statistically significant additional stability in axial rotation ROM and in flexion NZ than ACDFP. The ACDFTP provided better stability than ACDFP in bending and axial rotation, and better stability than ACDFPW in bending for both ROM and NZ. There was no significant difference in extension with either ROM or NZ for the three fixation methods. CONCLUSIONS: The spines treated with ACDFTP demonstrated the most effective stabilization, followed by those treated with ACDFPW, and then ACDFP. The spines receiving ACDFP also revealed a higher stability than the intact spine in most loading modes; thus ACDFP can also provide a relatively effective stabilization in bilateral cervical facet dislocation, but with the aid of a brace.

Mechanical offloading of incisional wounds is associated with transcriptional downregulation of inflammatory pathways in a large animal model.

Cutaneous scarring is a major source of morbidity and current therapies to mitigate scar formation remain ineffective. Although wound fibrosis and inflammation are highly linked, only recently have mechanical forces been implicated in these pathways. Our group has developed a topical polymer device that significantly reduces post-injury scar formation via the manipulation of mechanical forces. Here we extend these studies to examine the genomewide transcriptional effects of mechanomodulation during scar formation using a validated large animal model, the red Duroc pig. We demonstrate that mechanical loading of incisional wounds upregulates expression of genes associated with inflammatory and fibrotic pathways, and that device-mediated offloading of these wounds reverses these effects. Validation studies are needed to clarify the clinical significance of these findings.

Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling.

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Mesenchymal stem cells can participate in ischemic neovascularization.

BACKGROUND: Cells from the bone marrow contribute to ischemic neovascularization, but the identity of these cells remains unclear. The authors identify mesenchymal stem cells as a bone marrow-derived progenitor population that is able to engraft into peripheral tissue in response to ischemia. METHODS: A murine model of skin ischemia was used. Bone marrow, blood, and skin were harvested at different time points and subjected to flow cytometric analysis for mesenchymal and hematopoietic markers (n = 3 to 7 per time point). Using a parabiotic model pairing donor green fluorescent protein (GFP)-positive with recipient wild-type mice, progenitor cell engraftment was examined in ischemic tissue by fluorescence microscopy, and engrafted cells were analyzed by flow cytometry for endothelial and mesenchymal markers. In vitro, the ability of both bone marrow- and adipose-derived mesenchymal stem cells to adopt endothelial characteristics was examined by analyzing (1) the ability of mesenchymal stem cells to take up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and (2) phenotypic changes of mesenchymal stem cells co-cultured with GFP-labeled endothelial cells or under hypoxic/vascular endothelial growth factor stimulation. RESULTS: In vivo, the bone marrow mesenchymal stem cell population decreased significantly immediately after surgery, with subsequent engraftment of these cells in ischemic tissue. Engrafted cells lacked the panhematopoietic antigen CD45, consistent with a mesenchymal origin. In vitro, bone marrow- and adipose-derived mesenchymal stem cells took up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and expressed endothelial markers under hypoxic conditions. CONCLUSIONS: The authors' data suggest that mesenchymal precursor cells can give rise to endothelial progenitors. Consequently, cell-based therapies augmenting the mesenchymal stem cell population could represent powerful alternatives to current therapies for ischemic vascular disease.

Prospective comparison of topical moxifloxacin in eliminating conjunctival bacterial flora following a one-day or one-hour application.

PURPOSE: The aim of this study was to compare the efficacy of a 1-hour(h) versus 1-day application of topical moxifloxacin in eliminating conjunctival bacterial flora. METHODS: In this prospective, nonrandomized, controlled trial, the surgical eyes of 60 patients scheduled for intraocular surgery received topical moxifloxacin four times a day, starting 1 day prior to surgery and three additional applications at 5-minute intervals 1 h before surgery. The nonsurgical eye of each patient only received three applications of the same antibiotic at 5-minute intervals 1 h before surgery. Conjunctival cultures were obtained at baseline and after antibiotic application. RESULTS: Prior to antibiotic application, 80% of surgical eyes and 70% of nonsurgical eyes had positive cultures. Following the 1-day application, significantly fewer eyes (40%) had positive cultures (P < 0.0001), with a further reduction to 32% with three additional doses 1 h prior to surgery. In the nonsurgical eye, the decrease in the percentage of positive cultures, from 55% to 53% following the three applications 1 h prior to surgery, was not significant (P > 0.9999). The 1-day application was associated with significantly fewer positive cultures, compared to the 1-h group (P = 0.0267). CONCLUSIONS: The one-day application of moxifloxacin resulted in significantly fewer positive conjunctival cultures, compared with a 1-h application.

Comparison of the biomechanical stability of dense cancellous allograft with tricortical iliac autograft and fibular allograft for cervical interbody fusion.

Several choices are available for cervical interbody fusion after anterior cervical discectomy. A recent option is dense cancellous allograft (CS) which is characterized by an open-matrix structure that may promote vascularization and cellular penetration during early osseous integration. However, the biomechanical stability of CS should be comparable to that of the tricortical iliac autograft (AG) and fibular allograft (FA) to be an acceptable alternative to these materials. The purpose of this study was to compare the initial biomechanical stability of CS to that of AG and FA in a one-level anterior cervical discectomy and interbody fusion (ACDF) model. Twelve human cervical spines (C3-T1) were loaded in six modes of motion and evaluated under three conditions: (1) intact, (2) after ACDF using CS, AG, and FA in alternating sequences, and (3) after ACDF with anterior plating. Three reflective markers were placed on the adjacent vertebral bodies. Intervertebral motion was measured with a video-based motion-capture system (MacReflex, Qualisys, Sweden). Torques were applied to a maximum of 2.0 N m. The range-of-motion and neutral-zone values measured in each loading mode were compared. No graft material displayed significant differences in biomechanical stability in any of the tested loading modes, suggesting that the initial stability of CS is comparable to that of AG and FA. Anterior cervical plating significantly increased biomechanical stability in all modes.