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Liu K, Fu XW, Wang ZM. Msx1-Modified Rat Bone Marrow Mesenchymal Stem Cell Therapy for Rotator Cuff Repair: A Comprehensive Analysis of Tendon-Bone Healing and Cellular Mechanisms. J Orthop Res 2025; 43:859-869. [PMID: 39739627 DOI: 10.1002/jor.26039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Revised: 11/01/2024] [Accepted: 12/02/2024] [Indexed: 01/02/2025]
Abstract
This study investigates the therapeutic potential of Msx1-overexpressing bone marrow mesenchymal stem cells (BMSCs) in enhancing tendon-bone healing in rotator cuff injuries. BMSCs were genetically modified to overexpress Msx1 and were evaluated in vitro for their proliferation, migration, and differentiation potential. Results demonstrated that Msx1 overexpression significantly increased BMSC proliferation and migration while inhibiting osteogenic and chondrogenic differentiation. In a rat model of acute rotator cuff injury, Msx1-BMSCs embedded in a hydrogel scaffold were implanted at the tendon-bone junction. Micro-CT analysis revealed substantial new bone formation in the Msx1-BMSC group, and histological evaluation showed organized collagen and cartilage structures at the repair site. Biomechanical testing further confirmed enhanced structural strength in the Msx1-BMSC-treated group. These findings suggest that Msx1 modification enhances BMSC-mediated repair by promoting cell proliferation and migration, facilitating tendon-bone integration. This Msx1-based approach presents a promising strategy for advancing regenerative therapies for rotator cuff injuries.
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Affiliation(s)
- Kang Liu
- Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xia-Wei Fu
- Department of Orthopaedic Surgery, First Affiliated Hospital of Navy Medical University (Changhai Hospital), Shanghai, China
| | - Zi-Min Wang
- Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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2
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Nurhidayat L, Benes V, Blom S, Gomes I, Firdausi N, de Bakker MAG, Spaink HP, Richardson MK. Tokay gecko tail regeneration involves temporally collinear expression of HOXC genes and early expression of satellite cell markers. BMC Biol 2025; 23:6. [PMID: 39780185 PMCID: PMC11715542 DOI: 10.1186/s12915-024-02111-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2024] [Accepted: 12/27/2024] [Indexed: 01/11/2025] Open
Abstract
BACKGROUND Regeneration is the replacement of lost or damaged tissue with a functional copy. In axolotls and zebrafish, regeneration involves stem cells produced by de-differentiation. These cells form a growth zone which expresses developmental patterning genes at its apex. This system resembles an embryonic developmental field where cells undergo pattern formation. Some lizards, including geckos, can regenerate their tails, but it is unclear whether they show a "development-like" regeneration pathway. RESULTS Using the tokay gecko (Gekko gecko) model species, we examined seven stages of tail regeneration, and three stages of embryonic tail bud development, using transcriptomics, single-cell sequencing, and in situ hybridization. We find no apical growth zone in the regenerating tail. The transcriptomes of the regenerating vs. embryonic tails are quite different with respect to developmental patterning genes. Posterior HOXC genes were activated in a temporally collinear sequence in the regenerating tail. The major precursor populations were stromal cells (regenerating tail) vs. pluripotent stem cells (embryonic tail). Segmented skeletal muscles were regenerated with no expression of classical segmentation genes, but with the early activation of satellite cell markers. CONCLUSIONS Our study suggests that tail regeneration in the tokay gecko-unlike tail development-might rely on the activation of resident stem cells, guided by pre-existing positional information.
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Affiliation(s)
- Luthfi Nurhidayat
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
- Faculty of Biology, Universitas Gadjah Mada, Jalan Teknika Selatan Sekip Utara, Yogyakarta, 55281, Indonesia
| | - Vladimir Benes
- Genomics Core Facility, European Molecular Biology Laboratory Heidelberg, Meyerhofstraße 1, Heidelberg, 69117, Germany
| | - Sira Blom
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
| | - Inês Gomes
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
| | - Nisrina Firdausi
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
| | - Merijn A G de Bakker
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
| | - Herman P Spaink
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands
| | - Michael K Richardson
- Institute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.
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Tsadaris SA, Komatsu DE, Grubisic V, Ramos RL, Hadjiargyrou M. A GCaMP reporter mouse with chondrocyte specific expression of a green fluorescent calcium indicator. Bone 2024; 188:117234. [PMID: 39147354 PMCID: PMC11392458 DOI: 10.1016/j.bone.2024.117234] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 08/08/2024] [Accepted: 08/11/2024] [Indexed: 08/17/2024]
Abstract
One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca++) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca++-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca++ binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations.
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Affiliation(s)
- Sotirios A Tsadaris
- Department of Biological & Chemical Sciences, New York Institute of Technology, Old Westbury, NY, USA
| | - David E Komatsu
- Department of Orthopaedics and Rehabilitation, Stony Brook University, Stony Brook, NY, USA
| | - Vladimir Grubisic
- Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, USA; Center for Biomedical Innovation, College of Osteopathic Medicine, New York Institute of Technology, USA
| | - Raddy L Ramos
- Department of Biomedical Sciences, College of Osteopathic Medicine, New York Institute of Technology, USA
| | - Michael Hadjiargyrou
- Department of Biological & Chemical Sciences, New York Institute of Technology, Old Westbury, NY, USA.
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Hadjiargyrou M, Kotsiopriftis M, Lauzier D, Hamdy RC, Kloen P. Activation of Wnt signaling in human fracture callus and nonunion tissues. Bone Rep 2024; 22:101780. [PMID: 39005846 PMCID: PMC11245924 DOI: 10.1016/j.bonr.2024.101780] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/30/2024] [Revised: 06/07/2024] [Accepted: 06/18/2024] [Indexed: 07/16/2024] Open
Abstract
The Wnt signaling pathway is a key molecular process during fracture repair. Although much of what we now know about the role of this pathway in bone is derived from in vitro and animal studies, the same cannot be said about humans. As such, we hypothesized that Wnt signaling will also be a key process in humans during physiological fracture healing as well as in the development of a nonunion (hypertrophic and oligotrophic). We further hypothesized that the expression of Wnt-signaling pathway genes/proteins would exhibit a differential expression pattern between physiological fracture callus and the pathological nonunion tissues. We tested these two hypotheses by examining the mRNA levels of key Wnt-signaling related genes: ligands (WNT4, WNT10a), receptors (FZD4, LRP5, LRP6), inhibitors (DKK1, SOST) and modulators (CTNNB1 and PORCN). RNA sequencing from calluses as well as from the two nonunion tissue types, revealed that all of these genes were expressed at about the same level in these three tissue types. Further, spatial expression experiments identified the cells responsible of producing these proteins. Robust expression was detected in osteoblasts for the majority of these genes except SOST which displayed low expression, but in contrast, was mostly detected in osteocytes. Many of these genes were also expressed by callus chondrocytes as well. Taken together, these results confirm that Wnt signaling is indeed active during both human physiological fracture healing as well as in pathological nonunions.
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Affiliation(s)
- Michael Hadjiargyrou
- Department of Biological & Chemical Sciences, New York Institute of Technology, Old Westbury, NY 11568, USA
| | - Maria Kotsiopriftis
- Division of Orthopaedic Surgery, Shriners Hospital for Children, Montreal Children Hospital, McGill University, Montreal, QC H4A 0A9, Canada
| | - Dominique Lauzier
- Division of Orthopaedic Surgery, Shriners Hospital for Children, Montreal Children Hospital, McGill University, Montreal, QC H4A 0A9, Canada
| | - Reggie C Hamdy
- Division of Orthopaedic Surgery, Shriners Hospital for Children, Montreal Children Hospital, McGill University, Montreal, QC H4A 0A9, Canada
| | - Peter Kloen
- Department of Orthopedic Surgery and Sports Medicine, Amsterdam UMC, location Meibergdreef 9, Amsterdam, the Netherlands
- Amsterdam Movement Sciences, (Tissue Function and Regeneration), Amsterdam, the Netherlands
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Martin A, Kawaguchi R, Wang Q, Salusky IB, Pereira RC, Wesseling-Perry K. Chromatin accessibility and epigenetic deoxyribose nucleic acid (DNA) modifications in chronic kidney disease (CKD) osteoblasts: a study of bone and osteoblasts from pediatric patients with CKD. JBMR Plus 2024; 8:ziad015. [PMID: 38694428 PMCID: PMC11059997 DOI: 10.1093/jbmrpl/ziad015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Revised: 01/17/2023] [Accepted: 12/01/2023] [Indexed: 05/04/2024] Open
Abstract
Maturation defects are intrinsic features of osteoblast lineage cells in CKD patients. These defects persist ex vivo, suggesting that CKD induces epigenetic changes in bone cells. To gain insights into which signaling pathways contribute to CKD-mediated, epigenetically driven, impairments in osteoblast maturation, we characterized RNA expression and DNA methylation patterns by RNA-Seq and MethylationEpic in primary osteoblasts from nine adolescent and young adult dialysis patients with end-stage kidney disease and three healthy references. ATAC-Seq was also performed on a subset of osteoblasts. Bone matrix protein expression was extracted from the iliac crest and evaluated by proteomics. Gene set enrichment analysis was used to establish signaling pathways consistently altered in chromatin accessibility, DNA methylation, and RNA expression patterns. Single genes were suppressed in primary osteoblasts using shRNA and mineralization characterized in vitro. The effect of nuclear factor of activated T cells (NFAT) signaling suppression was also assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) incorporation. We found that signaling pathways critical for osteoblast differentiation were strongly downregulated in CKD osteoblasts. Gene set enrichment analysis identified highly significant methylation changes, differential chromatin accessibility, and altered RNA expression in NFAT signaling targets. NFAT inhibition reduced osteoblast proliferation. Combined analysis of osteoblast RNA expression and whole bone matrix composition identified 13 potential ligand-receptor pairs. In summary, epigenetic changes in CKD osteoblasts associate with altered expression of multiple osteoblast genes and signaling pathways. An increase in NFAT signaling may play a role in impaired CKD osteoblast maturation. Epigenetic changes also associate with an altered bone matrix, which may contribute to bone fragility. Further studies are necessary to elucidate the pathways affected by these genetic alterations since elucidating these pathways will be vital to correcting the underlying biology of bone disease in the CKD population.
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Affiliation(s)
- Aline Martin
- Division of Nephrology and Hypertension, Center for Translational Metabolism and Health, Feinberg Cardiovascular and Renal Research Institute, Northwestern University, Evanston, IL 60208
| | - Riki Kawaguchi
- Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095
- David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA 90095
| | - Qing Wang
- Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095
- David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA 90095
| | - Isidro B Salusky
- Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095
| | - Renata C Pereira
- Department of Pediatrics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095
| | - Katherine Wesseling-Perry
- Division of Nephrology, Department of Pediatrics, The University of Arizona, Phoenix Children’s Hospital, Phoenix, AZ 850156
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Hadjiargyrou M, Salichos L, Kloen P. Identification of the miRNAome in human fracture callus and nonunion tissues. J Orthop Translat 2023; 39:113-123. [PMID: 36909863 PMCID: PMC9996375 DOI: 10.1016/j.jot.2023.01.005] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2022] [Revised: 01/09/2023] [Accepted: 01/31/2023] [Indexed: 03/14/2023] Open
Abstract
Background Nonunions remain a challenging post-traumatic complication that often leads to a financial and health burden that affects the patient's quality of life. Despite a wealth of knowledge about fracture repair, especially gene and more recently miRNA expression, much remains unknown about the molecular differences between normal physiological repair (callus tissue) and a nonunion. To probe this lack of knowledge, we embarked on a study that sought to identify and compare the human miRNAome of normal bone to that present in a normal fracture callus and those from two different classic nonunion types, hypertrophic and oligotrophic. Methods Normal bone and callus tissue samples were harvested during revision surgery from patients with physiological fracture repair and nonunions (hypertrophic and oligotrophic) and analyzed using histology. Also, miRNAs were isolated and screened using microarrays followed by bioinformatic analyses, including, differential expression, pathways and biological processes, as well as elucidation of target genes. Results Out of 30,424 mature miRNAs (from 203 organisms) screened via microarrays, 635 (∼2.1%) miRNAs were found to be upregulated and 855 (∼2.8%) downregulated in the fracture callus and nonunion tissues as compared to intact bone. As our tissue samples were derived from humans, we focused on the human miRNAs and out of the 4223 human miRNAs, 86 miRNAs (∼2.0%) were upregulated and 51 (∼1.2%) were downregulated. Although there were similarities between the three experimental samples, we also found specific miRNAs that were unique to individual samples. We further identified the predicted target genes from these differentially expressed miRNAs as well as the relevant biological processes, including specific signaling pathways that are activated in all three experimental samples. Conclusion Collectively, this is the first comprehensive study reporting on the miRNAome of intact bone as compared to fracture callus and nonunion tissues. Further, we identify specific miRNAs involved in normal physiological fracture repair as well as those of nonunions. The translational potential of this article The data generated from this study further increase our molecular understanding of the roles of miRNAs during normal and aberrant fracture repair and this knowledge can be used in the future in the development of miRNA-based therapeutics for skeletal regeneration.
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Affiliation(s)
- Michael Hadjiargyrou
- Department of Biological & Chemical Sciences, New York Institute of Technology, Old Westbury, NY, 11568, USA
| | - Leonidas Salichos
- Department of Biological & Chemical Sciences, New York Institute of Technology, Old Westbury, NY, 11568, USA
| | - Peter Kloen
- Department of Orthopedic Surgery and Sports Medicine, Amsterdam UMC Location Meibergdreef, Amsterdam, the Netherlands
- Amsterdam Movement Sciences, (Tissue Function and Regeneration), Amsterdam, the Netherlands
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Mollentze J, Durandt C, Pepper MS. An In Vitro and In Vivo Comparison of Osteogenic Differentiation of Human Mesenchymal Stromal/Stem Cells. Stem Cells Int 2021; 2021:9919361. [PMID: 34539793 PMCID: PMC8443361 DOI: 10.1155/2021/9919361] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 07/23/2021] [Accepted: 08/20/2021] [Indexed: 12/11/2022] Open
Abstract
The use of stem cells in regenerative medicine, including tissue engineering and transplantation, has generated a great deal of enthusiasm. Mesenchymal stromal/stem cells (MSCs) can be isolated from various tissues, most commonly, bone marrow but more recently adipose tissue, dental pulp, and Wharton's jelly, to name a few. MSCs display varying phenotypic profiles and osteogenic differentiating capacity depending and their site of origin. MSCs have been successfully differentiated into osteoblasts both in vitro an in vivo but discrepancies exist when the two are compared: what happens in vitro does not necessarily happen in vivo, and it is therefore important to understand why these differences occur. The osteogenic process is a complex network of transcription factors, stimulators, inhibitors, proteins, etc., and in vivo experiments are helpful in evaluating the various aspects of this osteogenic process without distractions and confounding variables. With that in mind, the results of in vitro experiments need to be carefully considered and interpreted with caution as they do not perfectly replicate the conditions found within living organisms. This is where in vivo experiments help us better understand interactions that might occur in the osteogenic process that cannot be replicated in vitro. Potentially, these differences could also be exploited to develop an optimal MSC cell therapeutic product that can be used for bone disorders. There are many bone disorders, most of which cause a great deal of discomfort. Clinically acceptable protocols could be developed in which MSCs are used to aid in bone regeneration providing relief for patients with chronic pain. The aim of this review is to examine the differences between studies conducted in vitro and in vivo with regard to the osteogenic process to better define the gaps in current osteogenic research. By better understanding osteogenic differentiation, we can better define treatment strategies for various bone disorders.
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Affiliation(s)
- Jamie Mollentze
- Institute for Cellular and Molecular Medicine, Department of Immunology; SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
| | - Chrisna Durandt
- Institute for Cellular and Molecular Medicine, Department of Immunology; SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
| | - Michael S. Pepper
- Institute for Cellular and Molecular Medicine, Department of Immunology; SAMRC Extramural Unit for Stem Cell Research and Therapy, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
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Inoue S, Takito J, Nakamura M. Site-Specific Fracture Healing: Comparison between Diaphysis and Metaphysis in the Mouse Long Bone. Int J Mol Sci 2021; 22:ijms22179299. [PMID: 34502206 PMCID: PMC8430651 DOI: 10.3390/ijms22179299] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 08/24/2021] [Accepted: 08/25/2021] [Indexed: 12/14/2022] Open
Abstract
The process of fracture healing varies depending upon internal and external factors, such as the fracture site, mode of injury, and mechanical environment. This review focuses on site-specific fracture healing, particularly diaphyseal and metaphyseal healing in mouse long bones. Diaphyseal fractures heal by forming the periosteal and medullary callus, whereas metaphyseal fractures heal by forming the medullary callus. Bone healing in ovariectomized mice is accompanied by a decrease in the medullary callus formation both in the diaphysis and metaphysis. Administration of estrogen after fracture significantly recovers the decrease in diaphyseal healing but fails to recover the metaphyseal healing. Thus, the two bones show different osteogenic potentials after fracture in ovariectomized mice. This difference may be attributed to the heterogeneity of the skeletal stem cells (SSCs)/osteoblast progenitors of the two bones. The Hox genes that specify the patterning of the mammalian skeleton during embryogenesis are upregulated during the diaphyseal healing. Hox genes positively regulate the differentiation of osteoblasts from SSCs in vitro. During bone grafting, the SSCs in the donor’s bone express Hox with adaptability in the heterologous bone. These novel functions of the Hox genes are discussed herein with reference to the site-specificity of fracture healing.
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Signaling Pathway and Transcriptional Regulation in Osteoblasts during Bone Healing: Direct Involvement of Hydroxyapatite as a Biomaterial. Pharmaceuticals (Basel) 2021; 14:ph14070615. [PMID: 34206843 PMCID: PMC8308723 DOI: 10.3390/ph14070615] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Revised: 06/19/2021] [Accepted: 06/23/2021] [Indexed: 02/07/2023] Open
Abstract
Bone defects and periodontal disease are pathological conditions that may become neglected diseases if not treated properly. Hydroxyapatite (HA), along with tricalcium phosphate and bioglass ceramic, is a biomaterial widely applied to orthopedic and dental uses. The in vivo performance of HA is determined by the interaction between HA particles with bone cells, particularly the bone mineralizing cells osteoblasts. It has been reported that HA-induced osteoblastic differentiation by increasing the expression of osteogenic transcription factors. However, the pathway involved and the events that occur in the cell membrane have not been well understood and remain controversial. Advances in gene editing and the discovery of pharmacologic inhibitors assist researchers to better understand osteoblastic differentiation. This review summarizes the involvement of extracellular signal-regulated kinase (ERK), p38, Wnt, and bone morphogenetic protein 2 (BMP2) in osteoblastic cellular regulation induced by HA. These advances enhance the current understanding of the molecular mechanism of HA as a biomaterial. Moreover, they provide a better strategy for the design of HA to be utilized in bone engineering.
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10
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Komatsu DE, Duque E, Hadjiargyrou M. MicroRNAs and fracture healing: Pre-clinical studies. Bone 2021; 143:115758. [PMID: 33212318 PMCID: PMC7769985 DOI: 10.1016/j.bone.2020.115758] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2020] [Revised: 11/13/2020] [Accepted: 11/13/2020] [Indexed: 12/28/2022]
Abstract
During the past several years, pre-clinical experiments have established that microRNAs (miRNAs), small non-coding RNAs, serve as key regulatory molecules of fracture healing. Their easy modulation with agonists and antagonists make them highly desirable targets for future therapeutic strategies, especially for pathophysiologic fractures that either do not heal (nonunions) or are delayed. It is now well documented that these problematic fractures lead to human suffering and impairment of life quality. Additionally, financial difficulties are also encountered as work productivity decreases and income is reduced. Moreover, targeting miRNAs may also be an avenue to enhancing normal physiological fracture healing. Herein we present the most current knowledge of the involvement of miRNAs during fracture healing in pre-clinical studies. Following a brief description on the nature of miRNAs and of the fracture healing process, we present data from studies focusing specifically, on miRNA regulation of osteoblast differentiation and osteogenesis (within the context of known signaling pathways), chondrocytes, angiogenesis, and apoptosis, all critical to successful bone repair. Further, we also discuss miRNAs and exosomes. We hope that this manuscript serves as a comprehensive review that will facilitate basic/translational scientists in the orthopaedic arena to realize and further decipher the biological and future therapeutic impact of these small regulatory RNA molecules, especially as they relate to the molecular events of each of the major phases of fracture healing.
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Affiliation(s)
- David E Komatsu
- Department of Orthopaedics and Rehabilitation, Stony Brook University, United States of America
| | - Edie Duque
- Department of Orthopaedics and Rehabilitation, Stony Brook University, United States of America
| | - Michael Hadjiargyrou
- Department of Biological and Chemical Sciences, New York Institute of Technology, United States of America.
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11
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Owen A, Newsome PN. Mesenchymal Stromal Cells, a New Player in Reducing Complications From Liver Transplantation? Front Immunol 2020; 11:1306. [PMID: 32636850 PMCID: PMC7318292 DOI: 10.3389/fimmu.2020.01306] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Accepted: 05/22/2020] [Indexed: 12/12/2022] Open
Abstract
In response to the global burden of liver disease there has been a commensurate increase in the demand for liver transplantation. However, due to a paucity of donor organs many centers have moved toward the routine use of marginal allografts, which can be associated with a greater risk of complications and poorer clinical outcomes. Mesenchymal stromal cells (MSC) are a multi-potent progenitor cell population that have been utilized to modulate aberrant immune responses in acute and chronic inflammatory conditions. MSC exert an immunomodulatory effect on innate and adaptive immune systems through the release of both paracrine soluble factors and extracellular vesicles. Through these routes MSC can switch the regulatory function of the immune system through effects on macrophages and T regulatory cells enabling a switch of phenotype from injury to restoration. A key benefit seems to be their ability to tailor their response to the inflammatory environment without compromising the host ability to fight infection. With over 200 clinical trials registered to examine MSC therapy in liver disease and an increasing number of trials of MSC therapy in solid organ transplant recipients, there is increasing consideration for their use in liver transplantation. In this review we critically appraise the potential role of MSC therapy in the context of liver transplantation, including their ability to modulate reperfusion injury, their role in the reduction of medium term complications in the biliary tree and their potential to enhance tolerance in transplanted organs.
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Affiliation(s)
- Andrew Owen
- National Institute for Health Research Birmingham, Biomedical Research Centre at University Hospitals Birmingham NHS Foundation Trust, University of Birmingham, Birmingham, United Kingdom.,Department of Anesthesia and Critical Care, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom
| | - Philip N Newsome
- National Institute for Health Research Birmingham, Biomedical Research Centre at University Hospitals Birmingham NHS Foundation Trust, University of Birmingham, Birmingham, United Kingdom.,Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom.,Liver Unit, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom
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12
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Mastrolia I, Foppiani EM, Murgia A, Candini O, Samarelli AV, Grisendi G, Veronesi E, Horwitz EM, Dominici M. Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review. Stem Cells Transl Med 2019; 8:1135-1148. [PMID: 31313507 PMCID: PMC6811694 DOI: 10.1002/sctm.19-0044] [Citation(s) in RCA: 203] [Impact Index Per Article: 33.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Accepted: 06/17/2019] [Indexed: 02/06/2023] Open
Abstract
Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC‐tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage‐restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. stem cells translational medicine2019;8:1135–1148
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Affiliation(s)
- Ilenia Mastrolia
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
| | - Elisabetta Manuela Foppiani
- Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University Department of Pediatrics, Atlanta, Georgia, USA
| | - Alba Murgia
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
| | | | - Anna Valeria Samarelli
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
| | - Giulia Grisendi
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
| | - Elena Veronesi
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.,Technopole of Mirandola TPM, Mirandola, Modena, Italy
| | - Edwin M Horwitz
- Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta and Emory University Department of Pediatrics, Atlanta, Georgia, USA
| | - Massimo Dominici
- Laboratory of Cellular Therapy, Program of Cell Therapy and Immuno-Oncology, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.,Rigenerand srl, Medolla, Modena, Italy.,Technopole of Mirandola TPM, Mirandola, Modena, Italy
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13
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Barton-Owen TB, Szabó R, Somorjai IML, Ferrier DEK. A Revised Spiralian Homeobox Gene Classification Incorporating New Polychaete Transcriptomes Reveals a Diverse TALE Class and a Divergent Hox Gene. Genome Biol Evol 2018; 10:2151-2167. [PMID: 29986009 PMCID: PMC6118893 DOI: 10.1093/gbe/evy144] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/06/2018] [Indexed: 11/13/2022] Open
Abstract
The diversity of mechanisms and capacity for regeneration across the Metazoa present an intriguing challenge in evolutionary biology, impacting on the burgeoning field of regenerative medicine. Broad taxonomic sampling is essential to improve our understanding of regeneration, and studies outside of the traditional model organisms have proved extremely informative. Within the historically understudied Spiralia, the Annelida have an impressive variety of tractable regenerative systems. The biomeralizing, blastema-less regeneration of the head appendage (operculum) of the serpulid polychaete keelworm Spirobranchus (formerly Pomatoceros) lamarcki is one such system. To profile potential regulatory mechanisms, we classified the homeobox gene content of opercular regeneration transcriptomes. As a result of retrieving several difficult-to-classify homeobox sequences, we performed an extensive search and phylogenetic analysis of the TALE and PRD-class homeobox gene content of a broad selection of lophotrochozoan genomes. These analyses contribute to our increasing understanding of the diversity, taxonomic extent, rapid evolution, and radical flexibility of these recently discovered homeobox gene radiations. Our expansion and integration of previous nomenclature systems helps to clarify their cryptic orthology. We also describe an unusual divergent S. lamarcki Antp gene, a previously unclassified lophotrochozoan orphan gene family (Lopx), and a number of novel Nk class orphan genes. The expression and potential involvement of many of these lineage- and clade-restricted homeobox genes in S. lamarcki operculum regeneration provides an example of diversity in regenerative mechanisms, as well as significantly improving our understanding of homeobox gene evolution.
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Affiliation(s)
- Thomas B Barton-Owen
- Gatty Marine Laboratory, The Scottish Oceans Institute, School of Biology, University of St. Andrews, United Kingdom
- The Biomedical Sciences Research Complex, School of Biology, University of St. Andrews, United Kingdom
| | - Réka Szabó
- Gatty Marine Laboratory, The Scottish Oceans Institute, School of Biology, University of St. Andrews, United Kingdom
| | - Ildiko M L Somorjai
- Gatty Marine Laboratory, The Scottish Oceans Institute, School of Biology, University of St. Andrews, United Kingdom
- The Biomedical Sciences Research Complex, School of Biology, University of St. Andrews, United Kingdom
| | - David E K Ferrier
- Gatty Marine Laboratory, The Scottish Oceans Institute, School of Biology, University of St. Andrews, United Kingdom
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14
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Rux DR, Song JY, Pineault KM, Mandair GS, Swinehart IT, Schlientz AJ, Garthus KN, Goldstein SA, Kozloff KM, Wellik DM. Hox11 Function Is Required for Region-Specific Fracture Repair. J Bone Miner Res 2017; 32:1750-1760. [PMID: 28470721 PMCID: PMC5550340 DOI: 10.1002/jbmr.3166] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Revised: 04/26/2017] [Accepted: 05/01/2017] [Indexed: 12/23/2022]
Abstract
The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα+/CD51+/LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton. © 2017 American Society for Bone and Mineral Research.
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Affiliation(s)
- Danielle R. Rux
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Jane Y. Song
- Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Kyriel M. Pineault
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Gurjit S. Mandair
- Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA
| | - Ilea T. Swinehart
- Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Aleesa J. Schlientz
- Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Kayla N. Garthus
- Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Steve A. Goldstein
- Department of Orthopedic Surgery, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Ken M. Kozloff
- Department of Orthopedic Surgery, University of Michigan, Ann Arbor, MI 48109-2200, USA
| | - Deneen M Wellik
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-2200, USA
- Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, MI 48109-2200, USA
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15
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Rux DR, Wellik DM. Hox genes in the adult skeleton: Novel functions beyond embryonic development. Dev Dyn 2017; 246:310-317. [PMID: 28026082 DOI: 10.1002/dvdy.24482] [Citation(s) in RCA: 71] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Revised: 12/13/2016] [Accepted: 12/16/2016] [Indexed: 12/20/2022] Open
Abstract
Hox genes encode evolutionarily conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in regionally restricted domains and function to regulate the morphology of specific vertebral and long bone elements. Recent work has provided evidence that Hox genes continue to be regionally expressed in adult tissues. Fibroblasts cultured from adult tissues show broadly maintained Hox gene expression patterns. In the adult skeleton, Hox genes are expressed in progenitor-enriched populations of mesenchymal stem/stromal cells (MSCs), and genetic loss-of-function analyses have provided evidence that Hox genes function during the fracture healing process. This review will highlight our current understanding of Hox expression in the adult animal and its function in skeletal regeneration. Developmental Dynamics 246:310-317, 2017. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Danielle R Rux
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan
| | - Deneen M Wellik
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan.,Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan, Ann Arbor, Michigan
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16
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Camarata T, Vasilyev A, Hadjiargyrou M. Cloning of zebrafish Mustn1 orthologs and their expression during early development. Gene 2016; 593:235-241. [DOI: 10.1016/j.gene.2016.08.037] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Revised: 08/15/2016] [Accepted: 08/22/2016] [Indexed: 10/21/2022]
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17
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Hadjiargyrou M, Zhi J, Komatsu DE. Identification of the microRNA transcriptome during the early phases of mammalian fracture repair. Bone 2016; 87:78-88. [PMID: 27058875 DOI: 10.1016/j.bone.2016.03.011] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2016] [Revised: 03/10/2016] [Accepted: 03/22/2016] [Indexed: 10/22/2022]
Abstract
Fracture repair is a complex process that involves multiple biological processes requiring spatiotemporal expression of thousands of genes. The molecular regulation of this process is not completely understood. MicroRNAs (miRNAs) regulate gene expression by promoting mRNA degradation or blocking translation. To identify miRNAs expressed during fracture repair, we generated murine bone fractures and isolated miRNA-enriched RNA from intact and post-fracture day (PFD) 1, 3, 5, 7, 11, and 14 femurs. RNA samples were individually hybridized to mouse miRNA microarrays. Results indicated that 959 (51%) miRNAs were absent while 922 (49%) displayed expression in at least one sample. Of the 922 miRNAs, 306 (33.2%) and 374 (40.6%) were up- and down-regulated, respectively, in the calluses in comparison to intact bone. Additionally, 20 (2.2%) miRNAs displayed combined up- and down-regulated expression within the time course and the remaining 222 (24%) miRNAs did not exhibit any changes between calluses and intact bone. Quantitative-PCR validated the expression of several miRNAs. Further, we identified 2048 and 4782 target genes that were unique to the up- and down-regulated miRNAs, respectively. Gene ontology and pathway enrichment analyses indicated relevant biological processes. These data provide the first complete analysis of the miRNA transcriptome during the early phases of fracture repair.
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Affiliation(s)
- Michael Hadjiargyrou
- Department of Life Sciences, Theobald Science Center, Room 420, New York Institute of Technology, Old Westbury, NY 11568-8000, USA.
| | - Jizu Zhi
- Bioinformatics Core Facility, Stony Brook University, Stony Brook, NY 11794, USA.
| | - David E Komatsu
- Department of Orthopaedics, HSC T18 Room 85, Stony Brook University, Stony Brook, NY 11794-8181, USA.
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18
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de Jong DM, Seaver EC. A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta. PLoS One 2016; 11:e0149724. [PMID: 26894631 PMCID: PMC4764619 DOI: 10.1371/journal.pone.0149724] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 02/04/2016] [Indexed: 12/21/2022] Open
Abstract
Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.
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Affiliation(s)
- Danielle M. de Jong
- Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, Florida, United States of America
| | - Elaine C. Seaver
- Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, Florida, United States of America
- * E-mail:
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19
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Candini O, Spano C, Murgia A, Grisendi G, Veronesi E, Piccinno MS, Ferracin M, Negrini M, Giacobbi F, Bambi F, Horwitz EM, Conte P, Paolucci P, Dominici M. Mesenchymal progenitors aging highlights a miR-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis. Stem Cells 2015; 33:939-50. [PMID: 25428821 DOI: 10.1002/stem.1897] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Revised: 10/23/2014] [Accepted: 11/01/2014] [Indexed: 12/12/2022]
Abstract
Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR-196a upregulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence, and an improved osteogenesis linked to a dramatic increase of autocrine basic fibroblast growth factor secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance.
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Affiliation(s)
- Olivia Candini
- Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy
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20
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Seifert A, Werheid DF, Knapp SM, Tobiasch E. Role of Hox genes in stem cell differentiation. World J Stem Cells 2015; 7:583-595. [PMID: 25914765 PMCID: PMC4404393 DOI: 10.4252/wjsc.v7.i3.583] [Citation(s) in RCA: 107] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 11/20/2014] [Accepted: 12/17/2014] [Indexed: 02/06/2023] Open
Abstract
Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid of any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative medicine approaches.
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21
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González-Martín MC, Mallo M, Ros MA. Long bone development requires a threshold of Hox function. Dev Biol 2014; 392:454-65. [PMID: 24930703 DOI: 10.1016/j.ydbio.2014.06.004] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2013] [Revised: 05/30/2014] [Accepted: 06/04/2014] [Indexed: 11/30/2022]
Abstract
The Hoxd(Del(11-13)) mutant is one of the animal models for human synpolydactyly, characterized by short and syndactylous digits. Here we have characterized in detail the cartilage and bone defects in these mutants. We report two distinct phenotypes: (i) a delay and change in pattern of chondrocyte maturation of metacarpals/metatarsals and (ii) formation of a poor and not centrally positioned primary ossification center in the proximal-intermediate phalanx. In the metacarpals of Hoxd(Del(11-13)) mutants, ossification occurs postnataly, in the absence of significant Ihh expression and without the establishment of growth plates, following patterns similar to those of short bones. The strong downregulation in Ihh expression is associated with a corresponding increase of the repressor form of Gli3. To evaluate the contribution of this alteration to the phenotype, we generated double Hoxd(Del(11-13));Gli3 homozygous mutants. Intriguingly, these double mutants showed a complete rescue of the phenotype in metatarsals but only partial phenotypic rescue in metacarpals. Our results support Hox genes being required in a dose-dependent manner for long bone cartilage maturation and suggest that and excess of Gli3R mediates a significant part of the Hoxd(Del(11-13)) chondrogenic phenotype.
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Affiliation(s)
- Ma Carmen González-Martín
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), CSIC-Universidad de Cantabria-SODERCAN., 39011 Santander, Spain
| | - Moises Mallo
- Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal
| | - Marian A Ros
- Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), CSIC-Universidad de Cantabria-SODERCAN., 39011 Santander, Spain; Dpto. de Anatomía y Biología Celular, Universidad de Cantabria, 39011 Santander, Spain.
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22
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Liu X, Liao X, Luo E, Chen W, Bao C, Xu HHK. Mesenchymal stem cells systemically injected into femoral marrow of dogs home to mandibular defects to enhance new bone formation. Tissue Eng Part A 2014; 20:883-92. [PMID: 24125551 DOI: 10.1089/ten.tea.2012.0677] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Musculoskeletal diseases cost the U.S. $849 billion annually. To date, there has been no proof that remote long bone mesenchymal stem cells (BMSC) can home to craniofacial defects for bone regeneration. There has been no report that systemic BMSC injection can increase new bone formation in large animals. The objectives of this study were to use a sex-mismatched canine model for systemic BMSC injection and homing to mandibular defects and to investigate appendicular BMSC migration to craniofacial defects to increase new bone formation. Male beagle dog BMSC were injected into the femoral marrow cavity of female dogs upon which mandibular defects were created. The dogs were sacrificed at 6 weeks. Cells with Y chromosome markers were detected in defects of female dogs with systemic male BMSC injection, indicating the homing of the transplanted BMSC from femoral marrow to the mandibular defect. New bone formation in dogs with systemic BMSC injection was 20-40% higher than control without BMSC injection (p<0.05). Mineralized new bone percentage was increased by 20-40% due to systemic BMSC injection (p<0.05). In conclusion, this study proved that (1) allogeneic BMSC injected into long bone marrow are capable of homing to both appendicular and craniofacial bone in large animals and (2) systemically injected BMSC can significantly increase new bone formation in dog's mandibular defects. These results may help advance the understanding of stem cell homing and present a therapy to enhance bone repair, which may have a wide applicability to the regenerative medicine field.
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Affiliation(s)
- Xian Liu
- 1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University , Chengdu, China
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23
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Xiao SM, Gao Y, Cheung CL, Bow CH, Lau KS, Sham PC, Tan KCB, Kung AWC. Association of CDX1 binding site of periostin gene with bone mineral density and vertebral fracture risk. Osteoporos Int 2012; 23:1877-87. [PMID: 22215184 PMCID: PMC3368110 DOI: 10.1007/s00198-011-1861-1] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2011] [Accepted: 09/12/2011] [Indexed: 12/03/2022]
Abstract
SUMMARY Periostin (POSTN) as a regulator of osteoblast differentiation and bone formation may affect susceptibility to osteoporosis. This study suggests POSTN as a candidate gene for bone mineral density (BMD) variation and vertebral fracture risk, which could better our understanding about the genetic pathogenesis of osteoporosis and will be useful in clinic in the future. INTRODUCTION The genetic determination of osteoporosis is complex and ill-defined. Periostin (POSTN), an extracellular matrix secreted by osteoblasts and a regulator of osteoblast differentiation and bone formation, may affect susceptibility to osteoporosis. METHODS We adopted a tag-single nucleotide polymorphism (SNP) based association method followed by imputation-based verification and identification of a causal variant. The association was investigated in 1,572 subjects with extreme-BMD and replicated in an independent population of 2,509 subjects. BMD was measured by dual X-ray absorptiometry. Vertebral fractures were identified by assessing vertebral height from X-rays of the thoracolumbar spine. Association analyses were performed with PLINK toolset and imputation analyses with MACH software. The top imputation finding was subsequently validated by genotyping. Interactions between POSTN and another BMD-related candidate gene sclerostin (SOST) were analyzed using MDR program and validated by logistical regression analyses. The putative transcription factor binding with target sequence was confirmed by electrophoretic mobility shift assay (EMSA). RESULTS Several SNPs of POSTN were associated with BMD or vertebral fractures. The most significant polymorphism was rs9547970, located at the -2,327 bp upstream (P = 6.8 × 10(-4)) of POSTN. Carriers of the minor allele G per copy of rs9547970 had 1.33 higher risk of vertebral fracture (P = 0.007). An interactive effect between POSTN and SOST upon BMD variation was suggested (P < 0.01). A specific binding of CDX1 to the sequence of POSTN with the major allele A of rs9547970 but not the variant G allele was confirmed by EMSA. CONCLUSIONS Our results suggest POSTN as a candidate gene for BMD variation and vertebral fracture risk.
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Affiliation(s)
- S-M Xiao
- Department of Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
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24
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Hamada S, Satoh K, Hirota M, Kanno A, Umino J, Ito H, Masamune A, Kikuta K, Kume K, Shimosegawa T. The homeobox gene MSX2 determines chemosensitivity of pancreatic cancer cells via the regulation of transporter gene ABCG2. J Cell Physiol 2012; 227:729-38. [PMID: 21465479 DOI: 10.1002/jcp.22781] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Pancreatic cancer is one of the life-threatening cancers due to the difficulty in the curative surgery and resistance against conventional therapeutic strategies. Recent studies indicated that cancer stem cells, which exist as a small number of cells within the entire cancer tissue, contribute to the disease progression. Cancer stem cells reveal resistance against conventional chemotherapy, which is derived from the high-expression of multiple transporter genes. Our previous study demonstrated the aggravating role of the homeobox gene MSX2 as an inducer of epithelial-mesenchymal transition, and MSX2 turned out to correlate with the chemoresistance in the current study. Comprehensive analysis of the MSX2-target gene has identified ABCG2 as the responsible gene. Since previous studies reported the pivotal role of ABCG2 as a determining factor of cancer stem cells, the detailed regulatory mechanism of ABCG2 expression by MSX2 was investigated. As a result, the MSX2 expression level in each cell line well correlated with the ABCG2 expression level, and alteration of the MSX2 expression level by over-expression or siRNA-based knockdown affected the ABCG2 expression accordingly. Finally, we identified the functional cooperation of MSX2 and SP1 in the transcriptional regulation of ABCG2 via the SP1 binding elements within the ABCG2 promoter. These findings clarified the intriguing regulatory mechanism of the cancer stem cell-related gene, and will delineate a novel therapeutic target in pancreatic cancer.
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Affiliation(s)
- Shin Hamada
- Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai City, Miyagi, Japan
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Abstract
Reactive lesions of bone and soft tissue can appear alarming on histologic examination because they are often cellular and have atypical (activated) cytologic features, such as distinct nucleoli and mild hyperchromasia, and mitotic activity. Reactive lesions of bone and periosteum also produce bone and cartilage matrix, resulting in confusion with osteosarcoma or chondrosarcoma. Careful attention to key cytomorphological features such as the pattern of bone formation, uniform appearance of cells, and absence of atypical mitoses should help identify the reactive nature of a lesion. Correlation with clinical and radiological findings is also imperative to avoid misclassification of the tumor because reactive lesions often arise at sites where osteosarcoma and chondrosarcoma are rare (e.g., the hand) and lack aggressive radiological features. In this review we discuss reactive lesions of bone that are commonly confused with malignant neoplasms and that the practicing pathologist is likely to encounter at some point. Several of these lesions have had characteristic chromosomal translocations documented in recent years, but continue to be included as reactive lesions based on their overall clinicopathological features.
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Affiliation(s)
- Benjamin Hoch
- Department of Pathology, University of Washington Medical Center, Seattle, Washington 98195, USA.
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Wehrhan F, Hyckel P, Amann K, Ries J, Stockmann P, Schlegel K, Neukam F, Nkenke E. Msx-1 is suppressed in bisphosphonate-exposed jaw bone analysis of bone turnover-related cell signalling after bisphosphonate treatment. Oral Dis 2011; 17:433-42. [PMID: 21366807 DOI: 10.1111/j.1601-0825.2010.01778.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
OBJECTIVES Bone-destructive disease treatments include bisphosphonates and antibodies against receptor activator for nuclear factor κB ligand (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Aetiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonate-related ONJ. This study compared the expression of Msx-1, Bone Morphogenetic Protein (BMP)-2 and RANKL, in ONJ-affected and healthy jaw bone. MATERIAL AND METHODS An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n = 20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL and GAPDH mRNA levels. RESULTS Labelling indices were significantly lower for Msx-1 (P < 0.03) and RANKL (P < 0.003) and significantly higher (P < 0.02) for BMP-2 in ONJ compared with healthy bone. Expression was sevenfold lower (P < 0.03) for Msx-1, 22-fold lower (P < 0.001) for RANKL and eightfold higher (P < 0.02) for BMP-2 in ONJ bone. CONCLUSIONS Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonate-associated osteopetrosis and impaired bone remodelling in BP- and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explanation of the exclusivity of ONJ in jaw bone. Functional analyses of Msx-1- RANKL interaction during bone remodelling should be performed in the future.
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Affiliation(s)
- F Wehrhan
- Department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg, Erlangen, Germany.
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Wehrhan F, Hyckel P, Ries J, Stockmann P, Nkenke E, Schlegel KA, Neukam FW, Amann K. Expression of Msx-1 is suppressed in bisphosphonate associated osteonecrosis related jaw tissue-etiopathology considerations respecting jaw developmental biology-related unique features. J Transl Med 2010; 8:96. [PMID: 20942943 PMCID: PMC2973937 DOI: 10.1186/1479-5876-8-96] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2010] [Accepted: 10/13/2010] [Indexed: 01/06/2023] Open
Abstract
Background Bone-destructive disease treatments include bisphosphonates and antibodies against the osteoclast differentiator, RANKL (aRANKL); however, osteonecrosis of the jaw (ONJ) is a frequent side-effect. Current models fail to explain the restriction of bisphosphonate (BP)-related and denosumab (anti-RANKL antibody)-related ONJ to jaws. Msx-1 is exclusively expressed in craniofacial structures and pivotal to cranial neural crest (CNC)-derived periodontal tissue remodeling. We hypothesised that Msx-1 expression might be impaired in bisphosphonate-related ONJ. The study aim was to elucidate Msx-1 and RANKL-associated signal transduction (BMP-2/4, RANKL) in ONJ-altered and healthy periodontal tissue. Methods Twenty ONJ and twenty non-BP exposed periodontal samples were processed for RT-PCR and immunohistochemistry. An automated staining-based alkaline phosphatase-anti-alkaline phosphatase method was used to measure the stained cells:total cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed on ONJ-affected and healthy jaw periodontal samples (n = 20 each) to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels. Results Semi-quantitative assessment of the ratio of stained cells showed decreased Msx-1 and RANKL and increased BMP-2/4 (all p < 0.05) expression in ONJ-adjacent periodontal tissue. ONJ tissue also exhibited decreased relative gene expression for Msx-1 (p < 0.03) and RANKL (p < 0.03) and increased BMP-2/4 expression (p < 0.02) compared to control. Conclusions These results explain the sclerotic and osteopetrotic changes of periodontal tissue following BP application and substantiate clinical findings of BP-related impaired remodeling specific to periodontal tissue. RANKL suppression substantiated the clinical finding of impaired bone remodelling in BP- and aRANKL-induced ONJ-affected bone structures. Msx-1 suppression in ONJ-adjacent periodontal tissue suggested a bisphosphonate-related impairment in cellular differentiation that occurred exclusively jaw remodelling. Further research on developmental biology-related unique features of jaw bone structures will help to elucidate pathologies restricted to maxillofacial tissue.
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Affiliation(s)
- Falk Wehrhan
- Department of Oral and Maxillofacial Surgery University of Erlangen-Nuremberg, Germany.
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Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin remodeling factors. Mol Cell Biol 2010; 30:3531-41. [PMID: 20439491 DOI: 10.1128/mcb.00889-09] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblast-related genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis.
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Liu C, Gersch RP, Hawke TJ, Hadjiargyrou M. Silencing of Mustn1 inhibits myogenic fusion and differentiation. Am J Physiol Cell Physiol 2010; 298:C1100-8. [PMID: 20130207 DOI: 10.1152/ajpcell.00553.2009] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Mustn1 (Mustang, musculoskeletal temporally activated novel gene) was originally identified in fracture callus tissue, but its greatest expression is detected in skeletal muscle. Thus, we conducted experiments to investigate the expression and function of Mustn1 during myogenesis. Temporally, quantitative real-time PCR analysis of muscle samples from embryonic day 17 to 12 mo of age reveals that Mustn1 mRNA expression is greatest at 3 mo of age and beyond, consistent with the expression pattern of Myod. In situ hybridization shows abundant Mustn1 expression in somites and developing skeletal muscles, while in adult muscle, Mustn1 is localized to some peripherally located nuclei. Using RNA interference (RNAi), we investigated the function of Mustn1 in C2C12 myoblasts. Though silencing Mustn1 mRNA had no effect on myoblast proliferation, it did significantly impair myoblast differentiation, preventing myofusion. Specifically, when placed in low-serum medium for up to 6 days, Mustn1-silenced myoblasts elongated poorly and were mononucleated. In contrast, control RNAi-treated and parental myoblasts presented as large, multinucleated myotubes. Further supporting the morphological observations, immunocytochemistry of Mustn1-silenced cells demonstrated significant reductions in myogenin (Myog) and myosin heavy chain (Myhc) expression at 4 and 6 days of differentiation as compared with control and parental cells. The decreases in Myog and Myhc protein expression in Mustn1-silenced cells were associated with robust ( approximately 3-fold or greater) decreases in the expression of Myod and desmin (Des), as well as the myofusion markers calpain 1 (Capn1), caveolin 3 (Cav3), and cadherin 15 (M-cadherin; Cadh15). Overall, we demonstrate that Mustn1 is an essential regulator of myogenic differentiation and myofusion, and our findings implicate Myod and Myog as its downstream targets.
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Affiliation(s)
- Cheng Liu
- Dept. of Biomedical Engineering, Stony Brook Univ., NY 11794-2580, USA
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Houpis CH, Tosios KI, Papavasileiou D, Christopoulos PG, Koutlas IG, Sklavounou A, Alexandridis C. Parathyroid hormone-related peptide (PTHrP), parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1), and MSX1 protein are expressed in central and peripheral giant cell granulomas of the jaws. ACTA ACUST UNITED AC 2010; 109:415-24. [PMID: 20060342 DOI: 10.1016/j.tripleo.2009.09.026] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2009] [Revised: 09/08/2009] [Accepted: 09/18/2009] [Indexed: 01/07/2023]
Abstract
OBJECTIVE Parathyroid hormone-related peptide (PTHrP) binds to the parathyroid hormone receptor type 1 (PTHR1), which results in the activation of pathways in osteoblasts that promote osteoclastogenesis through the RANK/RANKL system. RANK/RANKL expression has been shown in central giant cell granuloma of the jaws but PTHrP/PTHR1 has not. MSX1 protein is a classical transcription regulator which promotes cell proliferation and inhibits cell differentiation by inhibiting master genes in tissues such as bone and muscle. It has been implicated in the pathogenesis of cherubism, and its expression has been reported in a single central giant cell granuloma (CGCG) case. We aimed, therefore, to study the expression of those proteins by the different cellular populations of central and peripheral giant cell granulomas (PGCGs) of the jaws. STUDY DESIGN Twenty cases of CGCG and 20 cases of PGCG of the jaws were retrospectively examined by immunohistochemistry for the percentage of positively staining cells to antibodies for PTHrP, PTHR1, and MSX1, using a semiquantitative method. RESULTS In both CGCG and PGCG of the jaws, PTHrP and PTHR1 were abundantly expressed by type I multinucleated giant cells (MGC) and mononucleated stromal cells (MSC) with vesicular nuclei, whereas type II MGC and MSC with pyknotic nuclei expressed those proteins to a lesser extent. In both CGCG and PGCG of the jaws, MSX1 was abundantly expressed by type I MGC and MSC but type II MGC did not express it. A statistically significant difference (P < .05) was observed between CGCG and PGCG in the expression of PTHrP in type II MGC and MSC with pyknotic nuclei and in the expression of PTHR1 in type II MGC. CONCLUSIONS We suggest that in CGCG and PGCG of the jaws, PTHrP-positive immature osteoblasts activate PTHR1-positive mature osteoblasts to produce RANKL which interacts with RANK on the PTHrP/PTHR1-positive osteoclast-precursor cells found in abundance in the stroma of giant cell lesions and induces osteoclastogenesis through the classic pathway. Cells of the jawbones, the periodontal ligament, or the dental follicle, originating from the neural crest, may be involved in the pathogenesis of giant cell lesions of the jaws. Further study is required for these suggestions to be proved.
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Affiliation(s)
- Constantinos H Houpis
- Department of Oral Pathology and Surgery, Dental School, National and Kapodestrian University of Athens, Athens, Greece.
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Adult rat bones maintain distinct regionalized expression of markers associated with their development. PLoS One 2009; 4:e8358. [PMID: 20027296 PMCID: PMC2792039 DOI: 10.1371/journal.pone.0008358] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2009] [Accepted: 11/21/2009] [Indexed: 12/31/2022] Open
Abstract
The incidence of limb bone fracture and subsequent morbidity and mortality due to excessive bone loss is increasing in the progressively ageing populations of both men and women. In contrast to bone loss in the weight-bearing limb, bone mass in the protective skull vault is maintained. One explanation for this could be anatomically diverse bone matrix characteristics generated by heterogeneous osteoblast populations. We have tested the hypothesis that adult bones demonstrate site-specific characteristics, and report differences at the organ, cell and transcriptome levels. Limb bones contain greater amounts of polysulphated glycosaminoglycan stained with Alcian Blue and have significantly higher osteocyte densities than skull bone. Site-specific patterns persist in cultured adult bone-derived cells both phenotypically (proliferation rate, response to estrogen and cell volumes), and at the level of specific gene expression (collagen triple helix repeat containing 1, reelin and ras-like and estrogen-regulated growth inhibitor). Based on genome-wide mRNA expression and cluster analysis, we demonstrate that bones and cultured adult bone-derived cells segregate according to site of derivation. We also find the differential expression of genes associated with embryological development (Skull: Zic, Dlx, Irx, Twist1 and Cart1; Limb: Hox, Shox2, and Tbx genes) in both adult bones and isolated adult bone-derived cells. Together, these site-specific differences support the view that, analogous to different muscle types (cardiac, smooth and skeletal), skull and limb bones represent separate classes of bone. We assign these differences, not to mode of primary ossification, but to the embryological cell lineage; the basis and implications of this division are discussed.
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High-density association study of 383 candidate genes for volumetric BMD at the femoral neck and lumbar spine among older men. J Bone Miner Res 2009; 24:2039-49. [PMID: 19453261 PMCID: PMC2791518 DOI: 10.1359/jbmr.090524] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Genetics is a well-established but poorly understood determinant of BMD. Whereas some genetic variants may influence BMD throughout the body, others may be skeletal site specific. We initially screened for associations between 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes and femoral neck and lumbar spine volumetric BMD (vBMD) measured from QCT scans among 862 community-dwelling white men >or=65 yr of age in the Osteoporotic Fractures in Men Study (MrOS). The most promising SNP associations (p < 0.01) were validated by genotyping an additional 1156 white men from MrOS. This analysis identified 8 SNPs in 6 genes (APC, DMP1, FGFR2, FLT1, HOXA, and PTN) that were associated with femoral neck vBMD and 13 SNPs in 7 genes (APC, BMPR1B, FOXC2, HOXA, IGFBP2, NFATC1, and SOST) that were associated with lumbar spine vBMD in both genotyping samples (p < 0.05). Although most associations were specific to one skeletal site, SNPs in the APC and HOXA gene regions were associated with both femoral neck and lumbar spine BMD. This analysis identifies several novel and robust genetic associations for volumetric BMD, and these findings in combination with other data suggest the presence of genetic loci for volumetric BMD that are at least to some extent skeletal-site specific.
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Gersch RP, Hadjiargyrou M. Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro. Bone 2009; 45:330-8. [PMID: 19410023 PMCID: PMC2706297 DOI: 10.1016/j.bone.2009.04.245] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/25/2009] [Revised: 03/24/2009] [Accepted: 04/22/2009] [Indexed: 12/01/2022]
Abstract
Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by approximately 2-6 fold, and reduced to approximately 32-52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of approximately 55-75%, and also approximately 34-40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.
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Affiliation(s)
- Robert P. Gersch
- Department of Biomedical Engineering, State University of New York, Stony Brook, Stony Brook, NY 11794
| | - Michael Hadjiargyrou
- Department of Biomedical Engineering, State University of New York, Stony Brook, Stony Brook, NY 11794
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Ackema KB, Charité J. Mesenchymal stem cells from different organs are characterized by distinct topographic Hox codes. Stem Cells Dev 2008; 17:979-91. [PMID: 18533811 DOI: 10.1089/scd.2007.0220] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Mesenchymal stem cells (MSC) are multipotent cells found as part of the stromal compartment of the bone marrow and in many other organs. They can be identified in vitro as CFU-F (colony forming unit-fibroblast) based on their ability to form adherent colonies of fibroblast-like cells in culture. MSC expanded in vitro retain characteristics appropriate to their tissue of origin. This is reflected in their propensity for differentiating towards specific lineages, and their capacity to generate, upon retransplantation in vivo, a stroma supporting typical lineages of hematopoietic cells. Hox genes encode master regulators of regional specification and organ development in the embryo and are widely expressed in the adult. We investigated whether they could be involved in determining tissue-specific properties of MSC. Hox gene expression profiles of individual CFU-F colonies derived from various organs and anatomical locations were generated, and the relatedness between these profiles was determined using hierarchical cluster analysis. This revealed that CFU-F have characteristic Hox expression signatures that are heterogeneous but highly specific for their anatomical origin. The topographic specificity of these Hox codes is maintained during differentiation, suggesting that they are an intrinsic property of MSC. Analysis of Hox codes of CFU-F from vertebral bone marrow suggests that MSC originate over a large part of the anterioposterior axis, but may not originate from prevertebral mesenchyme. These data are consistent with a role for Hox proteins in specifying cellular identity of MSC.
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Affiliation(s)
- Karin B Ackema
- Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands
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Khan SN, Solaris J, Ramsey KE, Yang X, Bostrom MP, Stephan D, Daluiski A. Identification of novel gene expression in healing fracture callus tissue by DNA microarray. HSS J 2008; 4:149-60. [PMID: 18752025 PMCID: PMC2553169 DOI: 10.1007/s11420-008-9087-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2008] [Accepted: 06/23/2008] [Indexed: 02/07/2023]
Abstract
Fracture healing requires controlled expression of thousands of genes. Only a small fraction of these genes have been isolated and fewer yet have been shown to play a direct role in fracture healing. The purpose of this study was threefold: (1) to develop a reproducible open femur model of fracture healing that produces consistent fracture calluses for subsequent RNA extraction, (2) to use this model to determine temporal expression patterns of known and unknown genes using DNA microarray expression profiling, and (3) to identify and validate novel gene expression in fracture healing. In the initial arm of the study, a total of 56 wild-type C57BL/6 mice were used. An open, stabilized diaphyseal femur fracture was created. Animals were killed at 1, 5, 7, 10, 14, 21, and 35 days after surgery and the femurs were harvested for analysis. At each time point, fractures were radiographed and sectioned for histologic analyses. Tissue from fracture callus at all stages following fracture yielded reproducibly large amounts of mRNA. Expression profiling revealed that genes cluster by function in a manner similar to the histologic stages of fracture healing. Based on the expression profiling of fracture tissue, temporal expression patterns of several genes known to be involved in fracture healing were verified. Novel expression of multiple genes in fracture callous tissue was also revealed including leptin and leptin receptor. In order to test whether leptin signaling is required for fracture repair, mice deficient in leptin or its receptor were fractured using the same model. Fracture calluses of mice deficient in both leptin or leptin receptor are larger than wild-type mice fractures, likely due to a delay in mineralization, revealing a previously unrecognized role of leptin signaling in fracture healing. This novel model of murine fracture repair is useful in examining both global changes in gene expression as well as individual signaling pathways, which can be used to identify specific molecular mechanisms of fracture healing.
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Affiliation(s)
- Safdar N. Khan
- Department of Orthopaedic Surgery, University of California, 4860 Y Street, Suite 1700, Davis, Sacramento, CA 95817 USA
| | - Jorge Solaris
- The Hospital for Special Surgery, 523 E 72nd Street, New York, NY 10021 USA
| | - Keri E. Ramsey
- Translational Genomics Research Institute, 445 N. Fifth Street, Phoenix, AZ 85004 USA
| | - Xu Yang
- The Hospital for Special Surgery, 523 E 72nd Street, New York, NY 10021 USA
| | | | - Dietrich Stephan
- Translational Genomics Research Institute, 445 N. Fifth Street, Phoenix, AZ 85004 USA
| | - Aaron Daluiski
- The Hospital for Special Surgery, 523 E 72nd Street, New York, NY 10021 USA
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Lien HC, Hsiao YH, Lin YS, Yao YT, Juan HF, Kuo WH, Hung MC, Chang KJ, Hsieh FJ. Molecular signatures of metaplastic carcinoma of the breast by large-scale transcriptional profiling: identification of genes potentially related to epithelial-mesenchymal transition. Oncogene 2007; 26:7859-71. [PMID: 17603561 DOI: 10.1038/sj.onc.1210593] [Citation(s) in RCA: 162] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Metaplastic carcinoma of the breast (MCB) is a poorly understood subtype of breast cancer. It is generally characterized by the coexistence of ductal carcinomatous and transdifferentiated sarcomatous components, but the underlying molecular alterations, possibly related to epithelial-mesenchymal transition (EMT), remain elusive. We performed transcriptional profiling using half-a-genome oligonucleotide microarrays to elucidate genetic profiles of MCBs and their differences to those of ductal carcinoma of breasts (DCBs) using discarded specimens of four MCBs and 34 DCBs. Unsupervised clustering disclosed distinctive expression profiles between MCBs and DCBs. Supervised analysis identified gene signatures discriminating MCBs from DCBs and between MCB subclasses. Notably, many of the discriminator genes were associated with downregulation of epithelial phenotypes and with synthesis, remodeling and adhesion of extracellular matrix, with some of them have known or inferred roles related to EMT. Importantly, several of the discriminator genes were upregulated in a mutant Snail-transfected MCF7 cell known to exhibit features of EMT, thereby indicating a crucial role for EMT in the pathogenesis of MCBs. Finally, the identification of SPARC and vimentin as poor prognostic factors reinforced the role of EMT in cancer progression. These data advance our understanding of MCB and offer clues to the molecular alterations underlying EMT.
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Affiliation(s)
- H C Lien
- Department of Pathology, College of Medicine, National Taiwan University, Taipei, Taiwan
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Komatsu DE, Bosch-Marce M, Semenza GL, Hadjiargyrou M. Enhanced bone regeneration associated with decreased apoptosis in mice with partial HIF-1alpha deficiency. J Bone Miner Res 2007; 22:366-74. [PMID: 17181398 PMCID: PMC2268762 DOI: 10.1359/jbmr.061207] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
UNLABELLED HIF-1alpha activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1alpha+/- fracture calluses are larger, stronger, and stiffer than HIF-1alpha+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. INTRODUCTION Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1alpha (HIF-1alpha), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1alpha is a key regulator of bone regeneration. MATERIALS AND METHODS Fixed femoral fractures were generated in mice with partial HIF-1alpha deficiency (HIF-1alpha+/-) and wildtype littermates (HIF-1alpha+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N=5-10) were subjected to microCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. RESULTS Surprisingly, fracture calluses from HIF-1alpha+/- mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1alpha+/+ calluses. Subsequent TUNEL staining showed that HIF-1alpha+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1alpha+/- calluses. CONCLUSIONS We conclude that partial HIF-1alpha deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.
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Affiliation(s)
- David E Komatsu
- Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, USA
| | - Marta Bosch-Marce
- Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Gregg L Semenza
- Vascular Biology Program, Institute for Cell Engineering, Department of Pediatrics, Medicine, Oncology, and Radiation Oncology and McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Michael Hadjiargyrou
- Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, USA
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Hassan MQ, Tare R, Lee SH, Mandeville M, Weiner B, Montecino M, van Wijnen AJ, Stein JL, Stein GS, Lian JB. HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes. Mol Cell Biol 2007; 27:3337-52. [PMID: 17325044 PMCID: PMC1899966 DOI: 10.1128/mcb.01544-06] [Citation(s) in RCA: 134] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
HOXA10 is necessary for embryonic patterning of skeletal elements, but its function in bone formation beyond this early developmental stage is unknown. Here we show that HOXA10 contributes to osteogenic lineage determination through activation of Runx2 and directly regulates osteoblastic phenotypic genes. In response to bone morphogenic protein BMP2, Hoxa10 is rapidly induced and functions to activate the Runx2 transcription factor essential for bone formation. A functional element with the Hox core motif was characterized for the bone-related Runx2 P1 promoter. HOXA10 also activates other osteogenic genes, including the alkaline phosphatase, osteocalcin, and bone sialoprotein genes, and temporally associates with these target gene promoters during stages of osteoblast differentiation prior to the recruitment of RUNX2. Exogenous expression and small interfering RNA knockdown studies establish that HOXA10 mediates chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation of these genes, correlating to active transcription. HOXA10 therefore contributes to early expression of osteogenic genes through chromatin remodeling. Importantly, HOXA10 can induce osteoblast genes in Runx2 null cells, providing evidence for a direct role in mediating osteoblast differentiation independent of RUNX2. We propose that HOXA10 activates RUNX2 in mesenchymal cells, contributing to the onset of osteogenesis, and that HOXA10 subsequently supports bone formation by direct regulation of osteoblast phenotypic genes.
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Affiliation(s)
- Mohammad Q Hassan
- Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA 01655-0106, USA
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Liu C, Hadjiargyrou M. Identification and characterization of the Mustang promoter: regulation by AP-1 during myogenic differentiation. Bone 2006; 39:815-24. [PMID: 16731063 DOI: 10.1016/j.bone.2006.04.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2006] [Revised: 04/04/2006] [Accepted: 04/08/2006] [Indexed: 11/19/2022]
Abstract
We previously identified Mustang (musculoskeletal temporally activated novel gene) with expression exclusively in the musculoskeletal system. Although its expression is almost undetectable in intact bone, it is robustly upregulated during bone regeneration. It is also abundantly expressed in adult skeletal muscle and tendon. As such, Mustang represents a marker for these cells and thus identifying its promoter would enable us to characterize its transcriptional regulation. To this end, we have isolated and characterized a 1512-bp mouse genomic clone representing the Mustang 5'-flanking region and identified a transcription start site, a TATA box, and multiple putative transcription factor binding sites (including AP-1 and AP-2). The activity of this promoter was detected in musculoskeletal cells and embryonic fibroblasts, even exceeding levels (145%) of the control SV40 promoter (in C2C12 cells). Further, the contribution of specific AP-1 and AP-2 sites was determined with serially deleted and mutated promoter constructs. Results indicate that one of the four AP-1 sites is required for substantial transcriptional activation, as its specific deletion or mutation decreases promoter activity by 32% and 40%, respectively. In contrast, deletion of both identified AP-2 sites results in only a 12% decrease in promoter activity. We further characterized the key AP-1 site by EMSA and determined that in both proliferating and differentiating C2C12 cells, only c-Fos, Fra-2 and JunD were required for transcriptional activation. Mustang's restricted tissue specificity and strong promoter makes this gene an ideal candidate for utilization in cell lineage studies that could unveil cellular/molecular mechanisms responsible for musculoskeletal development and regeneration.
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Affiliation(s)
- Cheng Liu
- Department of Biomedical Engineering, Stony Brook University, Psychology A Building, Room 338, Stony Brook, NY 11794-2580, USA
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Zhong N, Gersch RP, Hadjiargyrou M. Wnt signaling activation during bone regeneration and the role of Dishevelled in chondrocyte proliferation and differentiation. Bone 2006; 39:5-16. [PMID: 16459154 DOI: 10.1016/j.bone.2005.12.008] [Citation(s) in RCA: 99] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/10/2005] [Revised: 12/03/2005] [Accepted: 12/05/2005] [Indexed: 01/10/2023]
Abstract
Wnt signaling is intrinsically involved in diverse cellular activities during cell differentiation, early embryonic development and organogenesis. Although much is known regarding the effects of Wnt signaling in the developing skeletal system, its role during regeneration remains unclear. Herein, we show transcriptional activation of specific members and target genes of the Wnt signaling pathway. Specifically, all of the Wnt signaling members and target genes analyzed were found to be upregulated during the early stages of fracture repair, with the exception of LEF1 whose expression was downregulated. In addition, spatial expression analysis of Dishevelled (Dvl) and beta-catenin in the fracture callus revealed an identical pattern of expression with both proteins localizing in osteoprogenitor cells of the periosteum, osteoblasts and proliferating/pre-hypertrophic chondrocytes. Further, in vitro knockdown of all three Dvl isoforms in chondrocytes using small interfering RNAs (siRNA) leads to partial inhibition of cell proliferation and differentiation, decreased expression of chondrogenic markers (ColII, ColX, Sox9) and suppressed nuclear accumulation of unphosphorylated beta-catenin. Taken together, these data verify our previous finding that the Wnt signaling pathway is activated during bone regeneration, by characterizing the temporal and spatial expression of a broad spectrum of Wnt-signaling molecules. Our data also suggest that all three Dvl isoforms, acting through the Wnt canonical pathway, are critical regulatory molecules for chondrocyte proliferation and differentiation.
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Affiliation(s)
- Nan Zhong
- Department of Biomedical Engineering, State University of New York, Stony Brook, Psychology A Building, Room 338, Stony Brook, NY 11794-2580, USA
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Tsiridis E, Giannoudis PV. Transcriptomics and proteomics: advancing the understanding of genetic basis of fracture healing. Injury 2006; 37 Suppl 1:S13-9. [PMID: 16616752 DOI: 10.1016/j.injury.2006.02.036] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Fracture healing is a complex physiological post-natal process, which involves the coordination of several different cell types. Exploring the orchestration of events and the simultaneous activation of osteogenesis and chondrogenesis that recapitulates mammalian embryological skeletal development seems to be not only sophisticated but also challenging. A large number of genes involved in the above process are known, but many more remain to be discovered. The functional characterisation of these genes promises to elucidate the repair process as well as skeletal abnormalities and aging. We here review the current knowledge on early and late gene expression during fracture healing, the genes so far associated with osteoblast and osteoclast differentiation, the BMP antagonists, and the Wnts signalling pathway.
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Affiliation(s)
- Eleftherios Tsiridis
- Trauma & Orthopaedic Surgery, School of Medicine, University of Leeds, and St. James's University Hospital, Beckett Street, Leeds LS9 7TF, UK
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