Role of Matrix Metalloproteinase 13 in Both Endochondral and Intramembranous Ossification during Skeletal Regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research

The Role of Galectin-1 and Galectin-3 in the Mucosal Immune Response to Citrobacter rodentium Infection

Despite their abundance at gastrointestinal sites, little is known about the role of galectins in gut immune responses. We have therefore investigated the Citrobacter rodentium model of colonic infection and inflammation in Galectin-1 or Galectin-3 null mice. Gal-3 null mice showed a slight delay in colonisation after inoculation with C. rodentium and a slight delay in resolution of infection, associated with delayed T cell, macrophage and dendritic cell infiltration into the gut mucosa. However, Gal-1 null mice also demonstrated reduced T cell and macrophage responses to infection. Despite the reduced T cell and macrophage response in Gal-1 null mice, there was no effect on C. rodentium infection kinetics and pathology. Overall, Gal-1 and Gal-3 play only a minor role in immunity to a gut bacterial pathogen

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β−Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (Mr = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate

Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development

BACKGROUND: During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton. METHODOLOGY/PRINCIPAL FINDINGS: To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants. CONCLUSIONS/SIGNIFICANCE: Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton

The Road Less Traveled: Regulation of Leukocyte Migration Across Vascular and Lymphatic Endothelium by Galectins

Leukocyte entry from the blood into inflamed tissues, exit into the lymphatics, and migration to regional lymph nodes are all crucial processes for mounting an effective adaptive immune response. Leukocytes must cross two endothelial cell layers, the vascular and the lymphatic endothelial cell layers, during the journey from the blood to the lymph node. The proteins and cellular interactions which regulate leukocyte migration across the vascular endothelium are well studied; however, little is known about the factors that regulate leukocyte migration across the lymphatic endothelium. Here, we will summarize evidence for a role for galectins, a family of carbohydrate-binding proteins, in regulating leukocyte migration across the vascular endothelium and propose that galectins are also involved in leukocyte migration across the lymphatic endothelium

Long term survival following the detection of circulating tumour cells in head and neck squamous cell carcinoma

Background Techniques for detecting circulating tumor cells in the peripheral blood of patients with head and neck cancers may identify individuals likely to benefit from early systemic treatment. Methods Reconstruction experiments were used to optimise immunomagnetic enrichment and RT-PCR detection of circulating tumor cells using four markers (ELF3, CK19, EGFR and EphB4). This method was then tested in a pilot study using samples from 16 patients with advanced head and neck carcinomas. Results Seven patients were positive for circulating tumour cells both prior to and after surgery, 4 patients were positive prior to but not after surgery, 3 patients were positive after but not prior to surgery and 2 patients were negative. Two patients tested positive for circulating cells but there was no other evidence of tumor spread. Given this patient cohort had mostly advanced disease, as expected the detection of circulating tumour cells was not associated with significant differences in overall or disease free survival. Conclusion For the first time, we show that almost all patients with advanced head and neck cancers have circulating cells at the time of surgery. The clinical application of techniques for detection of spreading disease, such as the immunomagnetic enrichment RT-PCR analysis used in this study, should be explored further

Interplay of Nkx3.2, Sox9 and Pax3 Regulates Chondrogenic Differentiation of Muscle Progenitor Cells

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing