Bone Research www.nature.com/boneres ARTICLE OPEN The art of building bone: emerging role of chondrocyte-to- osteoblast transdifferentiation in endochondral ossification Patrick Aghajanian1 and Subburaman Mohan1,2,3,4 There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements (patterning phase) followed by their differentiation into cartilage (chondrocytes) or bone (osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of “transdifferentiation” per se is not recent, new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation (direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes. Bone Research (2018) 6:19 ; https://doi.org/10.1038/s41413-018-0021-z INTRODUCTION only use a small representative population. In the Drosophila The process of cell differentiation is a widely studied phenomenon intestine, Takashima et al. have shown that ectodermally derived which is the basis for all developmental processes. The basic hindgut cells migrate anteriorly to the midgut to form epithelial underlying principle of how cell differentiation proceeds is that cells of the endodermally derived midgut, becoming indistin- with each step of the differentiation pathway, cells become guishable from the surrounding epithelial cells.9 In zebrafish, programmed to follow a certain specified lineage progression during normal myocardial regeneration, Zhang et al. observed until they are terminally differentiated with an end point of that a population of atrial cardiomyocytes can migrate to the apoptosis or cell death.1–3 Recently, however, many studies have ventricle and repair it by direct atrial-to-ventricular transdiffer- introduced the idea of transdifferentiation, the differentiation of entiation and that this mechanism is regulated by notch cells (terminally differentiated or not) to a cell type that does not signaling.10 Not surprisingly, tissue regeneration in amphibians follow the normal, preprogrammed differentiation mechanism. and reptiles can also use transdifferentiation mechanisms. Transdifferentiation refers to a process where one mature cell Xenopus eye lenses, when removed, can regenerate as the result switches its phenotype and function to that of another mature of transdifferentiation of corneal epithelium to lens cells.11 Further differentiated cell type.4–8 This process occurs via two main studies have shown that upregulation of BMP and WNT12 as well mechanisms. The first is via direct transdifferentiation of one tissue as matrix metalloproteases13 are required for this direct type to another without undergoing an intermediate pluripotent transdifferentiation. state or becoming a progenitor cell, which will be denoted as Furthermore, there are examples of chondrogenic tissues direct transdifferentiation. The second major method occurs via an transdifferentiating into cell types of different origins and vice intermediate step, often manifested by a dedifferentiation and versa. Atherosclerotic lesions in mice have presented what seems redifferentiation. This mechanism will be denoted as intermediate to be the transdifferentiation of vascular smooth muscle to transdifferentiation. chondrogenic tissue. This was confirmed by the reduced Cell transdifferentiation has been described in the literature in expression of α-smooth muscle actin and the increased expression multiple tissue types and model organisms, and is therefore of SOX-9, a marker for immature chondrocytes, in the calcified neither a species- nor tissue-specific phenomenon. There are now lesions,14 as well as a different study that documented this event many examples of this phenomenon, but for this review, we will to occur via increased expression of tissue non-specific alkaline 1Musculoskeletal Disease Center, Veterans Affairs Loma Linda Healthcare System, Loma Linda, California, USA; 2Department of Medicine, Loma Linda University, Loma Linda, California, USA; 3Department of Orthopedics, Loma Linda University, Loma Linda, California, USA and 4Department of Biochemistry, Loma Linda University, Loma Linda, California, USA Correspondence: Subburaman Mohan ([email protected]) Received: 28 September 2017 Revised: 26 April 2018 Accepted: 2 May 2018 © The Author(s) 2018 The art of building bone: emerging role… P Aghajanian and S Mohan 2 1234567890();,: Fig. 1 Existing paradigm for long bone endochondral ossification. a Primary endochondral ossification begins with the formation of a chondrocyte template during embryogenesis. Chondrocytes undergo hypertrophy beginning from the mid-diaphysis, eventually extending to the epiphyseal poles. Vasculature invades the forming bone, transporting marrow, mesenchymal stromal cells, and osteoclasts. Hypertrophic cells undergo apoptosis, aided by the removal of matrix by osteoclasts. Mesenchymal stromal cells differentiate into osteoblasts and then osteocytes. b Secondary ossification occurs at the epiphysis post-natally in rodents. Immature chondrocytes at the center of the epiphysis become hypertrophic to produce mineralized collagen and eventually undergo apoptosis. Vasculature invades and transports marrow, mesenchymal stromal cells, and osteoclast precursors. Bone formation initiates at the center and extends peripherally. phosphatase and BMP-2 activation.15 In a reversal of roles, rat While there are many examples of transdifferentiation in the chondrocytes have been shown, when stimulated by neurogenic literature, this review will focus mainly on recent examples of growth factors (FGF-2, Neurobasal-A, EGF, and IGF-1), to transdif- transdifferentiation relating to the transformation of chondrogenic ferentiate into stellate neuronal cells with ablation of COL2 tissue into osteogenic tissue and the underlying mechanisms. expression and expression of neuron-specific proteins such as NF- 200, MAP-2, and β-III tubulin.16 Another case of osteogenic transdifferentiation involves the dedifferentiation of myoblasts via VASCULAR SOURCE OF OSTEOGENIC PRECURSORS IN THE BMP-2 induction of SMAD1, which is mediated by osteoactivin. CANONICAL PATHWAY OF ENDOCHONDRAL OSSIFICATION Osteoactivin, in turn, downregulates myogenic markers and To understand the different mechanisms of chondrocyte-to- upregulates osteogenic markers, such as RUNX2 and ALP.8,17–20 osteoblast transdifferentiation, it is first important to reference Finally, human gingivial fibroblasts have been shown, both in vitro the traditional canonical mechanism of endochondral ossifica- and in vivo to transdifferentiate to osteoblastic lineage cells when tion.23–28 Endochondral ossification, in the long bones, is the treated with 5-aza-2′-deoxycytidine followed by subsequent process of replacing cartilage with bone (Figs. 1 and 2a). This treatment with BMP2. This was confirmed in vitro by upregulation process differs in various bone tissues. In the long bones of Runx2 and Alp expression and in vivo subcutaneous transplan- themselves, there are different temporo-regional mechanisms of tation into mouse, which resulted in increased bone mineral endochondral ossification. These processes are referred to as content and bone volume/tissue volume.21 The issue of whether primary endochondral ossification (Fig. 1a) and secondary the transdifferentiation observed in some of these in vitro studies ossification (Fig. 1b). Primary ossification begins at embryonic can be attributed to the resident mesenchymal stem cells present day (E) 14.5–15.5 in rodents and encompasses the formation of in the cultures used remains to be examined. the periosteum, the bone collar (osteoid layer), and trabeculae Beyond these examples, it is important to note that any process which begins at the mid-diaphysis and extends to the growth of developing induced pluripotent stem cells from somatic cells is plates. Mechanistically, the most accepted mechanism for these a form of intermediate transdifferentiation. For example, in the models begins with the formation of a template of rapidly famous study by Takahashi and Yamanaka, mouse embryonic proliferating immature chondrocytes, which secrete a type 2 fibroblasts were transduced to express what are now known as collagen (COL2) matrix and make up both the perichondrium and the Yamanaka factors (Oct3/4, Sox2, c-Myc, Klf4) to induce a the immature chondrocytes of the diaphysis. This matrix is then pluripotent cell intermediate.