Controlled Delivery of Sdf-1 Alpha and Igf-1: Cxcr4(+) Cell Recruitment and Functional Skeletal Muscle Recovery

Therapeutic delivery of regeneration-promoting biological factors directly to the site of injury has demonstrated its efficacy in various injury models. Several reports describe improved tissue regeneration following local injection of tissue specific growth factors, cytokines and chemokines. Evidence exists that combined cytokine/growth factor treatment is superior for optimizing tissue repair by targeting different aspects of the regeneration response. The purpose of this study was to evaluate the therapeutic potential of the controlled delivery of stromal cell-derived factor-1alpha (SDF-1 alpha) alone or in combination with insulin-like growth factor-I (SDF-1 alpha/IGF-I) for the treatment of tourniquet-induced ischemia/reperfusion injury (TK-I/R) of skeletal muscle. We hypothesized that SDF-1 alpha will promote sustained stem cell recruitment to the site of muscle injury, while IGF-I will induce progenitor cell differentiation to effectively restore muscle contractile function after TK-I/R injury while concurrently reducing apoptosis. Utilizing a novel poly-ethylene glycol PEGylated fibrin gel matrix (PEG-Fib), we incorporated SDF-1 alpha alone (PEG-Fib/SDF-1 alpha) or in combination with IGF-I (PEG-Fib/SDF-1 alpha/IGF-I) for controlled release at the site of acute muscle injury. Despite enhanced cell recruitment and revascularization of the regenerating muscle after SDF-1 alpha treatment, functional analysis showed no benefit from PEG-Fib/SDF-1 alpha therapy, while dual delivery of PEG-Fib/SDF-1 alpha/IGF-I resulted in IGF-I-mediated improvement of maximal force recovery and SDF-1 alpha-driven in vivo neovasculogenesis. Histological data supported functional data, as well as highlighted the important differences in the regeneration process among treatment groups. This study provides evidence that while revascularization may be necessary for maximizing muscle force recovery, without modulation of other effects of inflammation it is insufficient.Kinesiology and Health Educatio

The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury

<div><p>Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized <i>in vitro</i> as well as <i>in vivo</i>. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived <i>in vitro</i> LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.</p></div

Analysis of the myofiber distribution 14 days post-reperfusion in GAS muscles treated with saline or 2x10⁶<i>in vitro</i> polarized macrophages 24h after TK-I/R injury.

<p><b>(A)</b> Representative H&E images of uninjured control GAS and injured, saline and MP treated GAS, at 14 days post-reperfusion; <b>(B) Top</b>: myofiber distribution in saline-treated group (black bars) relative to the contralateral uninjured control GAS(white bars); <b>Middle</b>: myofiber distribution after saline treatment (black bars) compared to M0 MP injected GAS (grey bars); <b>Bottom</b>: myofiber distribution following saline treatment (black bars) compared to M1(LPS/IFN-γ) MP treated GAS (pattern fill bars). n = 5/group; 3 fields of view/animal. Values expressed as mean ± SEM; (*) p<0.05 relative to saline; Student’s t-test.</p

<i>In vivo</i> quantification and characterization of MP populations 5 days after TK-I/R injury.

<p>A) Flow cytometric identification and quantification of myeloid and MP cell populations in TK-injured muscle. Total myeloid cells (CD45⁺CD11b⁺), resident MPs (CD11b⁺F4/80⁺PKH2.6^-), transplanted MPs (CD11b⁺F4/80⁺PKH2.6⁺).Values expressed as mean ±SEM, n = 3, B) Expression of CD206 and Ly-6C surface proteins on F4/80⁺MP populations in control and MP-treated muscles. Values expressed as mean ±SEM, n = 3.</p

Phenotypic analysis of <i>in vitro</i> polarized BM MPs.

<p>MPs were either left untreated (M0) or stimulated with 10ng/ml LPS/IFN-γ and IL-4/IL-13for 42 hours to induce classical (M1) and alternative (M2) activation phenotypes respectively. Flow cytometry was used to evaluate the expression of CD206 and Ly-6C surface proteins. <b>(A)</b> Representative plots of surface protein after <i>in vitro</i> MP polarization, <b>(B)</b> Mean fluorescence intensity of CD206 expression on the surface of polarized MPs, <b>(C)</b> Representative plot of PKH2.6 label and F4/80expression by <i>in vitro</i> polarized macrophages prior to transplantation.</p

Gastrocnemius muscle mass normalized to body weight and force recovery 14 days post-reperfusion after saline injection or the delivery of 2x10⁶<i>in vitro</i> polarized macrophages 24 h after TK-I/R injury.

<p><b>(A)</b> GAS mass(mg)/body weight (BW)(gm); <b>(B)</b> Force (N)/GAS mass (mg). Control n = 17, Saline n = 7, M0D1 (un-polarized) MPs n = 5, M1D1 (LPS/IFN-γ polarized (42h)) MPs n = 6. Values expressed as mean ± SEM. (*) p<0.05 relative to contralateral control; (#) p<0.05 relative to saline; (ƚ) p<0.05 relative to M0D1; one-way ANOVA, Tukey-HSD post-hoc.</p