Christopher R. Rathbone

Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway

Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1alpha activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program.

The promotion of a functional fibrosis in skeletal muscle with volumetric muscle loss injury following the transplantation of muscle-ECM

Tissue engineering strategies that primarily use biological extracellular matrices (ECMs) with or without the inclusion of a stem or progenitor cell source are under development for the treatment of trauma resulting in the loss of a large volume of skeletal muscle (i.e., volumetric muscle loss; VML). The explicit goal is to restore functional capacity to the injured tissue by promoting generation of muscle fibers. In the current study, a syngeneic muscle-derived ECM (mECM) was transplanted in a rat tibialis anterior (TA) muscle VML model. Instead of muscle fiber generation a large fibrotic mass was produced by mECM transplantation out to six months post-injury. Surprisingly, recovery of one-third of the original functional deficit was still achieved by two months post-injury following mECM transplantation. These counterintuitive findings may be due, at least in part, to the ability of mECM to attenuate muscle damage in the remaining muscle as compared to non-repaired muscle. These findings point to a novel role of biological ECMs for the treatment of VML, wherein the remaining muscle mass is protected from prolonged overload injury.

Autologous minced muscle grafts: a tissue engineering therapy for the volumetric loss of skeletal muscle

Volumetric muscle loss (VML) results in a large void deficient in the requisite materials for regeneration for which there is no definitive clinical standard of care. Autologous minced muscle grafts (MG), which contain the essential components for muscle regeneration, may embody an ideal tissue engineering therapy for VML. The purpose of this study was to determine if orthotopic transplantation of MG acutely after VML in the tibialis anterior muscle of male Lewis rats promotes functional tissue regeneration. Herein we report that over the first 16 wk postinjury, MG transplantation 1) promotes remarkable regeneration of innervated muscle fibers within the defect area (i.e., de novo muscle fiber regeneration); 2) reduced evidence of chronic injury in the remaining muscle mass compared with nonrepaired muscles following VML (i.e., transplantation attenuated chronically upregulated transforming growth factor-β1 gene expression and the presence of centrally located nuclei in 30% of fibers observed in nonrepaired muscles); and 3) significantly improves net torque production (i.e., ∼55% of the functional deficit in nonrepaired muscles was restored). Additionally, voluntary wheel running was shown to reduce the heightened accumulation of extracellular matrix deposition observed within the regenerated tissue of MG-repaired sedentary rats 8 wk postinjury (collagen 1% area: sedentary vs. runner, ∼41 vs. 30%), which may have been the result of an augmented inflammatory response [i.e., M1 (CCR7) and M2 (CD163) macrophage expression was significantly greater in runner than sedentary MG-repaired muscles 2 wk postinjury]. These findings support further exploration of autologous minced MGs for the treatment of VML.

Volumetric muscle loss leads to permanent disability following extremity trauma.

Extremity injuries comprise the majority of battlefield injuries and contribute the most to long-term disability of servicemembers. The purpose of this study was to better define the contribution of muscle deficits and volumetric muscle loss (VML) to the designation of long-term disability in order to better understand their effect on outcomes for limb-salvage patients. Medically retired servicemembers who sustained a combat-related type III open tibia fracture (Orthopedic cohort) were reviewed for results of their medical evaluation leading to discharge from military service. A cohort of battlefield-injured servicemembers (including those with nonorthopedic injuries) who were medically retired because of various injuries (General cohort) was also examined. Muscle conditions accounted for 65% of the disability of patients in the Orthopedic cohort. Among the General cohort, 92% of the muscle conditions were identified as VML. VML is a condition that contributes significantly to long-term disability, and the development of therapies addressing VML has the potential to fill a significant void in orthopedic care.

Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro

BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Skeletal muscle satellite cell activation following cutaneous burn in rats

BACKGROUND Cutaneous burn distant from skeletal muscles induces atrophy; however, its effect on muscle stem cells resident in skeletal muscle (satellite cells) distal to burn is not known. METHODS Satellite cell activation was measured in predominantly fast-twitch [tibialis anterior, extensor digitorum longus (EDL), plantaris, and gastrocnemius] and slow-twitch (soleus) muscles of rats that received either 40% total body surface area full-thickness scald burn or sham burn to the trunk area by determining bromodeoxyuridine incorporation, MyoD, and Pax7 immunohistochemistry in vivo ≤48 h after burn. To determine the effects of circulating factors on satellite cell activation, satellite cell cultures were treated with serum from sham or burn rats. RESULTS In vivo activation of satellite cells was increased in fast muscles isolated from burn as compared to sham animals, whereas a significant response was not seen in slow muscles. Serum taken from animals in the burn group increased the activation of satellite cells isolated from both sham and burn animals in vitro, suggesting that circulating factors have the potential to increase satellite cell activation following burn. CONCLUSIONS Increases in satellite cell activation in muscles distal to burn are fiber-type-dependent, and circulating factors may play a role in the activation of satellite cells following burn. A better understanding of the impact of burn on satellite cell functionality will allow us to identify the cellular mechanisms of long-term muscle atrophy.

Soluble Factors from Biofilms of Wound Pathogens Modulate Human Bone Marrow-derived Stromal Cell Differentiation, Migration, Angiogenesis, and Cytokine Secretion

BackgroundChronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro.ResultsExposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing.ConclusionsCollectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect.

Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro

Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9-12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.

Hypoxia Simultaneously Alters Satellite Cell-Mediated Angiogenesis and Hepatocyte Growth Factor Expression

Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell‐mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF‐deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF‐1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell‐mediated angiogenesis involves a reduction in satellite cell HGF expression. J. Cell. Physiol. 229: 572–579, 2014.

Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration

Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration.