Cbfb Regulates Bone Development by Stabilizing Runx Family Proteins

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA‐binding capacity. Cbfb‐deficient (Cbfb^?/?) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb^?/? mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb^fl/fl mice with Dermo1 Cre knock‐in mice. Cbfb^fl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb^fl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb^fl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfb^fl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb^fl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfb^fl/fl/Cre osteoblasts than Cbfb^fl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons. © 2014 American Society for Bone and Mineral Research.     

Runx2 Regulates Endochondral Ossification Through Control of Chondrocyte Proliferation and Differentiation

Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 gene in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2^ΔE8/ΔE8 mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C‐terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal levels of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth are disrupted in Runx2^ΔE8/ΔE8 mice. Loss of Runx2 in chondrocytes also impaired osteoprotegerin‐receptor activator of NF‐κB ligand (OPG‐RANKL) signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes, Gpr132, Sfn, c‐Myb, and Cyclin A1, to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes. © 2014 American Society for Bone and Mineral Research.     

Epiphyseal Chondrocyte Secondary Ossification Centers Require Thyroid Hormone Activation of Indian Hedgehog and Osterix Signaling

Thyroid hormones (THs) are known to regulate endochondral ossification during skeletal development via acting directly in chondrocytes and osteoblasts. In this study, we focused on TH effects on the secondary ossification center (SOC) because the time of appearance of SOCs in several species coincides with the time when peak levels of TH are attained. Accordingly, micro–computed tomography (µCT) evaluation of femurs and tibias at day 21 in TH‐deficient and control mice revealed that endochondral ossification of SOCs is severely compromised owing to TH deficiency and that TH treatment for 10 days completely rescued this phenotype. Staining of cartilage and bone in the epiphysis revealed that whereas all of the cartilage is converted into bone in the prepubertal control mice, this conversion failed to occur in the TH‐deficient mice. Immunohistochemistry studies revealed that TH treatment of thyroid stimulating hormone receptor mutant (Tshr^?/?) mice induced expression of Indian hedgehog (Ihh) and Osx in type 2 collagen (Col2)‐expressing chondrocytes in the SOC at day 7, which subsequently differentiate into type 10 collagen (Col10)/osteocalcin‐expressing chondro/osteoblasts at day 10. Consistent with these data, treatment of tibia cultures from 3‐day‐old mice with 10 ng/mL TH increased expression of Osx, Col10, alkaline phosphatase (ALP), and osteocalcin in the epiphysis by sixfold to 60‐fold. Furthermore, knockdown of the TH‐induced increase in Osx expression using lentiviral small hairpin RNA (shRNA) significantly blocked TH‐induced ALP and osteocalcin expression in chondrocytes. Treatment of chondrogenic cells with an Ihh inhibitor abolished chondro/osteoblast differentiation and SOC formation. Our findings indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix–producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes. © 2014 American Society for Bone and Mineral Research.     

Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry,Skeletal Development,and Osteoblast and Osteoclast Function

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild‐type or singly hemizygous animals. Cortical bone analysis by micro–computed tomography (μCT) revealed that 8‐week‐old male, compound Gja1^+/– Runx2^+/– mice have a marked increase in cross‐sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1^+/– Runx2^+/– mice closely approximate the cortical bone phenotypes seen in osteoblast‐specific Gja1‐conditional knockout models. Furthermore, μCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2‐day‐old Gja1^+/– Runx2^+/– neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2^+/– neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research     

Targeting of Mesenchymal Stromal Cells by Cre‐Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells

The targeting specificity of tissue‐specific Cre‐recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn‐Cre and Dmp1‐Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high‐resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12^gfp mice to identify Cxcl12‐abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn‐Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1‐Cre also targets a subset of CAR cells, in which expression of osteoblast‐lineage genes is enriched. Finally, we introduce a new tissue‐specific Cre‐recombinase, Tagln‐Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn‐Cre and Dmp1‐Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research.     

Positive Regulation of Adult Bone Formation by Osteoblast‐Specific Transcription Factor Osterix

Osterix (Osx) is essential for osteoblast differentiation and bone formation, because mice lacking Osx die within 1 h of birth with a complete absence of intramembranous and endochondral bone formation. Perinatal lethality caused by the disruption of the Osx gene prevents studies of the role of Osx in bones that are growing or already formed. Here, the function of Osx was examined in adult bones using the time‐ and site‐specific Cre/loxP system. Osx was inactivated in all osteoblasts by Col1a1‐Cre with the activity of Cre recombinase under the control of the 2.3‐kb collagen promoter. Even though no bone defects were observed in newborn mice, Osx inactivation with 2.3‐kb Col1a1‐Cre exhibited osteopenia phenotypes in growing mice. BMD and bone‐forming rate were decreased in lumbar vertebra, and the cortical bone of the long bones was thinner and more porous with reduced bone length. The trabecular bones were increased, but they were immature or premature. The expression of early marker genes for osteoblast differentiation such as Runx2, osteopontin, and alkaline phosphatase was markedly increased, but the late marker gene, osteocalcin, was decreased. However, no functional defects were found in osteoclasts. In summary, Osx inactivation in growing bones delayed osteoblast maturation, causing an accumulation of immature osteoblasts and reducing osteoblast function for bone formation, without apparent defects in bone resorption. These findings suggest a significant role of Osx in positively regulating osteoblast differentiation and bone formation in adult bone.     

Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

Effects of Deletion of ERα in Osteoblast‐Lineage Cells on Bone Mass and Adaptation to Mechanical Loading Differ in Female and Male Mice

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed‐background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell–specific ERαKO mice. Therefore, we crossed ERα floxed (ERα^fl/fl) and osteocalcin‐Cre (OC‐Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC‐ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro‐CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC‐ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC‐ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC‐ERαKO female L₅ vertebrae and with increased maximum moment in pOC‐ERαKO male femora. Female pOC‐ERαKO mice responded more to mechanical loading, whereas the response of pOC‐ERαKO male animals was similar to their littermate controls. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.     

Wdr5 is required for chick skeletal development

Wdr5, a bone morphogenetic protein 2 (BMP‐2)–induced protein belonging to the family of the WD repeat proteins, is expressed in proliferating and hypertrophic chondrocytes of the growth plate and in osteoblasts. Although previous studies have provided insight into the mechanisms by which Wdr5 affects chondrocyte and osteoblast differentiation, whether Wdr5 is required in vivo for endochondral bone development has not been addressed. In this study, using an avian replication competent retrovirus (RCAS) system delivering Wdr5 short hairpin (sh) RNA to silence Wdr5 in the developing limb, we report that reduction of Wdr5 levels delays endochondral bone development and consequently results in shortening of the skeletal elements. Shortening of the skeletal elements was due to impaired chondrocyte maturation, evidenced by a significant reduction of Runx2, type X collagen, and osteopontin expression. A decrease in Runx2, type collagen I, and ostepontin expression in osteoblasts and a subsequent defect in mineralized bone was observed as well when Wdr5 levels were reduced. Most important, retroviral misexpression of Runx2 rescued the phenotype induced by Wdr5 shRNA. These findings suggest that during limb development, Wdr5 is required for endochondral bone formation and that Wdr5 influences this process, at least in part, by regulating Runx2 expression. © 2010 American Society for Bone and Mineral Research.     

Localization of Pigment Epithelium-Derived Factor in Growing Mouse Bone

Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor found in a wide range of fetal and adult tissues, where it is thought to play a role in the regulation of angiogenesis during development. The temporal expression of PEDF during endochondral bone formation has not previously been reported. In this study, we analysed the expression pattern of PEDF in growing mouse hindlimbs from newborn day one through to maturation at week 9, using immunohistochemistry and in situ hybridization. PEDF expression was demonstrated in chondrocytes within the resting, proliferative and upper hypertrophic zones of the epiphyseal growth plate. The pattern of expression was consistent throughout the developmental stages of the mouse. In addition, PEDF was expressed by osteoblasts lining the bone spicules in the ossification zone of metaphyseal bone, as well as by osteoblasts lining cortical periosteum. These novel results demonstrate that PEDF is developmentally expressed in both cartilage and bone cells during endochondral bone formation, and strongly suggest that it may play a regulatory role in the processes of chondrocyte and osteoblast differentiation, endochondral ossification, and bone remodelling during growth and development of long bones.     

Loss of ephrinB1 in osteogenic progenitor cells impedes endochondral ossification and compromises bone strength integrity during skeletal development

The EphB receptor tyrosine kinase family and their ephrinB ligands have been implicated as mediators of skeletal development and bone homeostasis in humans, where mutations in ephrinB1 contribute to frontonasal dysplasia and coronal craniosynostosis. In mouse models, ephrinB1 has been shown to be a critical factor mediating osteoblast function. The present study examined the functional importance of ephrinB1 during endochondral ossification using the Cre recombination system with targeted deletion of ephrinB1 (EfnB1^fl/fl) in osteogenic progenitor cells, under the control of the osterix (Osx:Cre) promoter. The Osx:EfnB1^−/− mice displayed aberrant bone growth during embryonic and postnatal skeletal development up to 4 weeks of age, when compared to the Osx:Cre controls. Furthermore, compared to the Osx:Cre control mice, the Osx:EfnB1^−/− mice exhibited significantly weaker and less rigid bones, with a reduction in trabecular/ cortical bone formation, reduced trabecular architecture and a reduction in the size of the growth plates at the distal end of the femora from newborn through to 4 weeks of age. The aberrant bone formation correlated with increased numbers of tartrate resistant acid phosphatase positive osteoclasts and decreased numbers of bone lining osteoblasts in 4 week old Osx:EfnB1^−/− mice, compared to Osx:Cre control mice. Taken together, these observations demonstrate the importance of ephrinB1 signalling between cells of the skeleton required for endochondral ossification.     

异种脱钙骨基质、骨基质胶加释放系统移植的组织学和电镜观察

XDBM XBMG用小公牛长管状骨制备;释放系统由 CaSO_1、Na_2HPO_1、KH_2PO:(DSA）、Ca-Cl_2、Na_2HPO_4、KH_2PO_1(DSB)组成。Wistar大鼠24只分四组:(1)XDBM加DSA;(2)XDBM加DSB;(3)XBMG加DSA;(4)XBMG加DSB;不设对照。将四种移植物分别植入大鼠股四头肌肌穴。定期取材制片,TEM—100SX透射电镜观察。结果:2天巨噬细胞、淋巴细胞密集,细胞器发达,微绒毛状突起;4天间充质细胞贴附在XDBM、XBMG表面,细胞器发达伪足伸入移植物缝隙;8天成软骨细胞活跃,软骨细胞少;14天软骨细胞中部分细胞的细胞器固缩、溶解;22天网状骨小梁,骨细胞突起部分相互连接;34天新生骨渐成熟,骨髓细胞,个别哈氏管出现;几种生骨细胞和间充质细胞常在一个视野或网眼内,相贴很近,始终未发现破骨细胞。实验证明:XDBM、XBMG加DS诱导成骨的细胞来源是间充质细胞,直到34天仍见巨噬细胞存在尚未转化为破骨细胞。     

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFA^fl/fl) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFA^fl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFA^fl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFA^fl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.     

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR^f/y;Prx1‐Cre or ERα^f/f;Osx1‐Cre) or myeloid cell lineage (AR^f/y;LysM‐Cre or ERα^f/f;LysM‐Cre) and their descendants. Male AR^f/y;Prx1‐Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR^f/y;LysM‐Cre, ERα^f/f;Osx1‐Cre, or ERα^f/f;LysM‐Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR^f/y;Prx1‐Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts—not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s). © 2015 American Society for Bone and Mineral Research.     

Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are “borderline” chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single-cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell type–specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)-GFP⁺ osteoblasts and Cxcl12-GFP^high reticular stromal “CAR” cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.