In Vitro and in Vivo Endochondral Bone Formation Models Allow Identification of Anti-Angiogenic Compounds

Objective To support the preclinical evaluation of therapeutics that target chondrogenesis, our goal was to generate a rat strain that can noninvasively report endogenous chondrogenic activity. Design A transgene was constructed in which the dual expression of bioluminescent (firefly luciferase) and fluorescent (mCherry) reporters is controlled by regulatory sequences from rat Col2a1. Candidate lines were established on a Lewis background and characterized by serial bioluminescence imaging as well as ex vivo measurement of molecular reporter levels in several tissues. The sensitivity and specificity of the reporter strain were assessed in models of orthotopic and ectopic chondrogenesis. Results Substantial bioluminescence signal was detected from cartilaginous regions, including the appendicular synovial joints, spine, sternum, nose, and pinnae. Bioluminescent radiance was intense at 1 month of age, rapidly declined with continued development, yet remained detectable in 2-year-old animals. Explant imaging and immunohistochemistry confirmed that both molecular reporters were localized to cartilage. Implantation of wild-type bone marrow stromal cells into osteochondral defects made in both young adult and aged reporter rats led to a time-dependent elevation of intra-articular reporter activity concurrent with cartilaginous tissue repair. To stimulate ectopic, endochondral bone formation, bone morphogenetic protein 2 was overexpressed in the gastrocnemius muscle, which led to bioluminescent signal that closely preceded heterotopic ossification. Conclusions This strain can help develop strategies to stimulate cartilage repair and endochondral bone formation or to inhibit chondrogenesis associated with heterotopic ossification.

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Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.

The Journal of Cell Biology ◽

10.1083/jcb.126.6.1595 ◽

1994 ◽

Vol 126 (6) ◽

pp. 1595-1609 ◽

Cited By ~ 119

Author(s):

S E Gitelman ◽

M S Kobrin ◽

J Q Ye ◽

A R Lopez ◽

A Lee ◽

...

Keyword(s):

Bone Formation ◽

Tumor Cells ◽

Cho Cells ◽

Subcutaneous Tissue ◽

Endochondral Bone Formation ◽

Related Factor ◽

Tgf Beta ◽

Endochondral Bone ◽

Hypertrophic Cartilage

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta-related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation.

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Bone Marrow Mesenchymal Stem Cells Form Ectopic Woven Bone In Vivo Through Endochondral Bone Formation

Artificial Organs ◽

10.1111/j.1525-1594.2009.00728.x ◽

2009 ◽

Vol 33 (4) ◽

pp. 301-308 ◽

Cited By ~ 16

Author(s):

Sophia Chia-Ning Chang ◽

Ching-Lung Tai ◽

Hui-Ying Chung ◽

Tsung-Min Lin ◽

Long-Bin Jeng

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Woven Bone ◽

Marrow Mesenchymal Stem Cells

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Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model

Bone ◽

10.1016/s8756-3282(97)00180-4 ◽

1997 ◽

Vol 21 (5) ◽

pp. 385-392 ◽

Cited By ~ 77

Author(s):

V. Kartsogiannis ◽

J. Moseley ◽

B. McKelvie ◽

S.T. Chou ◽

D.K. Hards ◽

...

Keyword(s):

Bone Formation ◽

Rabbit Model ◽

Endochondral Bone Formation ◽

Temporal Expression ◽

Endochondral Bone ◽

Intramembranous Bone ◽

Intramembranous Bone Formation

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XBP1S Induces GEP and Enhances Endochondral Bone Growth

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.926-930.1136 ◽

2014 ◽

Vol 926-930 ◽

pp. 1136-1139

Author(s):

Feng Jin Guo ◽

Rong Jiang ◽

Xiao Feng Han

Keyword(s):

Growth Factor ◽

Bone Formation ◽

Bone Growth ◽

Ex Vivo ◽

Skeletal Development ◽

Endochondral Bone Formation ◽

Chondrocyte Differentiation ◽

Endochondral Bone ◽

Temporal And Spatial

We previously reported that transcription factor XBP1S is upregulated during chondrocyte differentiation and demonstrates the temporal and spatial expression pattern during skeletal development. Herein, we found that XBP1S stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo. In addition, XBP1S activates granulin-epithelin precursor (GEP), a growth factor known to stimulate chondrogenesis, then enhances GEP-stimulated chondrogenesis and endochondral bone formation. Collectively, these findings demonstrate that XBP1S positively regulates endochondral bone formation by activating GEP chondrogenic growth factor.

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Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.661989 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jessica Nulty ◽

Ross Burdis ◽

Daniel J. Kelly

Keyword(s):

Tissue Engineering ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Building Blocks ◽

Patient Specific ◽

Endochondral Bone Formation ◽

Hypertrophic Cartilage

Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue’s development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue “building blocks,” which could potentially be used in the treatment of challenging bone defects.

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A novel in vivo model to study endochondral bone formation; HIF-1α activation and BMP expression

Bone ◽

10.1016/j.bone.2006.08.005 ◽

2007 ◽

Vol 40 (2) ◽

pp. 409-418 ◽

Cited By ~ 42

Author(s):

Pieter J. Emans ◽

Frank Spaapen ◽

Don A.M. Surtel ◽

Keryn M. Reilly ◽

Andy Cremers ◽

...

Keyword(s):

Bone Formation ◽

In Vivo Model ◽

Endochondral Bone Formation ◽

Endochondral Bone

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Parathyroid Hormone–related Peptide (PTHrP)-dependent and -independent Effects of Transforming Growth Factor β (TGF-β) on Endochondral Bone Formation

The Journal of Cell Biology ◽

10.1083/jcb.145.4.783 ◽

1999 ◽

Vol 145 (4) ◽

pp. 783-794 ◽

Cited By ~ 121

Author(s):

Rosa Serra ◽

Andrew Karaplis ◽

Philip Sohn

Keyword(s):

Bone Formation ◽

Transforming Growth Factor ◽

Terminal Differentiation ◽

Transforming Growth Factor Β ◽

Endochondral Bone Formation ◽

Endochondral Bone ◽

Matrix Mineralization ◽

Parathyroid Hormone Related Peptide ◽

Independent Effects

Previously, we showed that expression of a dominant-negative form of the transforming growth factor β (TGF-β) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-β in limiting terminal differentiation in vivo. Parathyroid hormone–related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-β and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-β inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-β1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-β in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-β acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-β has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.

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