HOX11 Function in Musculoskeletal Development and Repair.

Previous genetic analyses of Hox loss-of-function phenotypes have demonstrated that these genes are essential regulators of skeletal patterning. However, as studies of Hox function in limb development have been limited to the skeleton, our understanding of the cell types in which Hox genes function and the tissues they pattern is incomplete. Utilizing a Hoxa11eGFP allele, we show that Hox11 is expressed in the connective tissue of the outer perichondrium, tendons and muscle connective tissue of the zeugopod region throughout all stages of development. Hox11 is not expressed in differentiated cartilage or bone, or in vascular or muscle cells in these regions. Hox11 genes provide patterning information to the muscle and tendon of the forelimb zeugopod in addition to patterning the skeletal elements. In Hox11 double mutants, numerous muscles and tendons of the forelimb zeugopod are absent and others fail to separate into properly patterned muscle bundles. Analyses of Hox11 compound mutants, in which three of for Hox11 alleles are mutant, demonstrate that muscle and tendon patterning is not secondary to skeletal malformations. Despite the normal skeletal phenotype of these embryos, significant disruption in patterning is observed. Recent evidence suggests that Hox expression is maintained throughout life and conserves aspects of the regionally restricted pattern observed during embryogenesis. Analysis of Hoxa11 expression at adult stages demonstrates that Hox11 remains expressed in skeletal tissues and is expressed in cells of the fracture callus during repair. Fracture healing is impaired in Hox11 compound mutants. Ulnar fractures in these animals show reduced cartilage formation, demonstrating that Hox11 genes are important for the early phases of bone repair. Thus, Hox genes are not simply regulators of skeletal morphology, but are key factors that regulate regional patterning and integration of the musculoskeletal system. Our results shift the paradigm of Hox function in establishing the body plan from key regulators that direct skeletal morphology to factors that control the patterning and integration of all three tissue types of the musculoskeletal system: muscle, tendon and bone. Our data also demonstrates a continued role for Hox genes throughout the life of the organism in skeletal repair and remodeling.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99922/1/swinehai_1.pd

Macrophage phenotype in response to ECM bioscaffolds

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ + LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers

The impact of detergents on the tissue decellularization process: a ToF-SIMS study

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy

Hox11 genes regulate postnatal longitudinal bone growth and growth plate proliferation

Hox genes are critical regulators of skeletal development and Hox9-13 paralogs, specifically, are necessary for appendicular development along the proximal to distal axis. Loss of function of both Hoxa11 and Hoxd11 results in severe malformation of the forelimb zeugopod. In the radius and ulna of these mutants, chondrocyte development is perturbed, growth plates are not established, and skeletal growth and maturation fails. In compound mutants in which one of the four Hox11 alleles remains wild-type, establishment of a growth plate is preserved and embryos develop normally through newborn stages, however, skeletal phenotypes become evident postnatally. During postnatal development, the radial and ulnar growth rate slows compared to wild-type controls and terminal bone length is reduced. Growth plate height is decreased in mutants and premature growth plate senescence occurs along with abnormally high levels of chondrocyte proliferation in the reserve and proliferative zones. Compound mutants additionally develop an abnormal curvature of the radius, which causes significant distortion of the carpal elements. The progressive bowing of the radius appears to result from physical constraint caused by the disproportionately slower growth of the ulna than the radius. Collectively, these data are consistent with premature depletion of forelimb zeugopod progenitor cells in the growth plate of Hox11 compound mutants, and demonstrate a continued function for Hox genes in postnatal bone growth and patterning