Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor–β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-β1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-β1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-β1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.

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Osteoblast AMP-Activated Protein Kinase Regulates Postnatal Skeletal Development in Male Mice

Endocrinology ◽

10.1210/en.2017-00357 ◽

2017 ◽

Vol 159 (2) ◽

pp. 597-608 ◽

Cited By ~ 8

Author(s):

Ippei Kanazawa ◽

Ayumu Takeno ◽

Ken-ichiro Tanaka ◽

Masakazu Notsu ◽

Toshitsugu Sugimoto

Keyword(s):

Protein Kinase ◽

Energy Homeostasis ◽

Bone Development ◽

Skeletal Development ◽

Formation Rate ◽

Osteoblastic Differentiation ◽

Bone Morphogenetic Protein 2 ◽

Bone Formation Rate ◽

Amp Activated Protein Kinase

Abstract Studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in bone development. Thus, to investigate in vivo roles of osteoblast AMPK, we conditionally inactivated Ampk in osterix (Osx)–expressing cells by crossing Osx-Cre mice with floxed AMPKα1 to generate mice lacking AMPKα1 in osteoblasts (Ampk−/− mice). Compared with wild-type and Ampk+/− mice, Ampk−/− mice displayed retardation of postnatal bone development, although bone deformity was not observed at birth. Microcomputed tomography showed significant reductions in trabecular bone volume, cortical bone length, and density, as well as increased cortical porosity in femur as well as development defects of skull in 8-week-old Ampk−/− mice. Surprisingly, histomorphometric analysis demonstrated that the number of osteoclasts was significantly increased, although bone formation rate was not altered. Loss of trabecular network connections and mass, as well as shortened growth plates and reduced thickness of cartilage adjacent to the growth plate, was observed in Ampk−/− mice. In primary cultured osteoblasts from calvaria, the expressions of alkaline phosphatase, type 1 collagen, osteocalcin, bone morphogenetic protein 2, Runx2, and osterix were significantly inhibited in Ampk−/− osteoblasts, whereas the expression of receptor activator of nuclear κB ligand (RANKL) and the RANKL/osteoprotegerin ratio were significantly increased. These findings indicate that osteoblastic AMPK plays important roles in bone development in vivo and that deletion of AMPK in osteoblasts decreases osteoblastic differentiation and enhances bone turnover by increasing RANKL expression.

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IGF-binding protein-5 stimulates osteoblast activity and bone accretion in ovariectomized mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.281.2.e283 ◽

2001 ◽

Vol 281 (2) ◽

pp. E283-E288 ◽

Cited By ~ 33

Author(s):

Dennis L. Andress

Keyword(s):

Bone Formation ◽

Binding Protein ◽

Formation Rate ◽

Bone Matrix ◽

Secretory Protein ◽

Mineral Density ◽

Bone Formation Rate ◽

Ovariectomized Mice ◽

Osteoblast Activity

Insulin-like growth factor binding protein-5 (IGFBP-5) is an osteoblast secretory protein that becomes incorporated into the mineralized bone matrix. In osteoblast cultures, IGFBP-5 stimulates cell proliferation by an IGF-independent mechanism. To evaluate whether IGFBP-5 can stimulate osteoblast activity and enhance bone accretion in a mouse model of osteoblast insufficiency, daily subcutaneous injections of either intact [IGFBP-5 (intact)] or carboxy-truncated IGFBP-5 [IGFBP-5-(1–169)] were given to ovariectomized (OVX) mice for 8 wk. Femur and spine bone mineral density (BMD), measured every 2 wk, showed early and sustained increases in response to IGFBP-5. Bone histomorphometry of cancellous bone showed significant elevations in the bone formation rate in both the femur metaphysis [IGFBP-5- (1)] only) and spine compared with OVX controls. IGFBP-5 also stimulated osteoblast number in the femur IGFBP-5-(1–169) only) and spine. These data indicate that IGFBP-5 effectively enhances bone formation and bone accretion in OVX mice by stimulating osteoblast activity. The finding that IGFBP-5-(1–169) is bioactive in vivo indicates that the carboxy-terminal portion is not required for this bone anabolic effect.

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Comparison of Release Profiles of Various Growth Factors from Biodegradable Carriers

MRS Proceedings ◽

10.1557/proc-530-13 ◽

1998 ◽

Vol 530 ◽

Cited By ~ 1

Author(s):

Y. Tabata ◽

M. Yamamoto ◽

Y. Ikada

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β1 ◽

The Body ◽

Bone Morphogenetic Protein 2 ◽

Biodegradable Hydrogel ◽

Tgf Β1

AbstractA biodegradable hydrogel was prepared by glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 as a carrier to release basic growth factors on the basis of polyion complexation. Basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein-2 (BMP-2) were sorbed from their aqueous solution into the dried gelatin hydrogels to prepare respective growth factor-incorporating hydrogels. Under an in vitro non-degradation condition, approximately 20 % of incorporated bFGF and TGF-β1 was released from the hydrogels within initial 40 min, followed by no further release, whereas a large initial release of BMP-2 was observed. After subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled growth factor in the mouse back, the remaining radioactivity was measured to estimate the in vivo release profile of growth factors. Incorporation into gelatin hydrogels enabled bFGF and TGF-β1 to retain in the body for about 15 days and the retention period well correlated with that of the gelatin hydrogel. Taken together, it is likely that the growth factors ionically complexed with acidic gelatin were released in vivo as a result of hydrogel biodegradation. On the contrary, basic BMP-2 did not ionically interact with acidic gelatin, resulting in no sustained released by the present biodegradable carrier system.

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Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair

10.1101/561837 ◽

2019 ◽

Author(s):

S. Herberg ◽

A. M. McDermott ◽

P. N. Dang ◽

D. S. Alt ◽

R. Tang ◽

...

Keyword(s):

Stem Cell ◽

Bone Formation ◽

Mechanical Loading ◽

Endochondral Ossification ◽

Bone Development ◽

Human Mesenchymal Stem Cell ◽

Bone Defects ◽

Mechanical Environment ◽

Tgf Β1

AbstractEndochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation and is directed by local morphogen signals and mechanical cues. Here, we aimed to mimic these developmental conditions for regeneration of large bone defects. We hypothesized that engineered human mesenchymal stem cell (hMSC) condensations with in situ presentation of transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles would promote endochondral regeneration of critical-sized rat femoral bone defects in a manner dependent on the in vivo mechanical environment. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with dual BMP-2 + TGF-β1 fully restoring mechanical bone function by week 12. In vivo ambulatory mechanical loading, initiated at week 4 by delayed unlocking of compliant fixation plates, significantly enhanced the bone formation rate in the four weeks after load initiation in the dual morphogen group. In vitro, local presentation of either BMP-2 alone or BMP-2 + TGF-β1 initiated endochondral lineage commitment of mesenchymal condensations, inducing both chondrogenic and osteogenic gene expression through SMAD3 and SMAD5 signaling. In vivo, however, endochondral cartilage formation was evident only in the BMP-2 + TGF-β1 group and was enhanced by mechanical loading. The degree of bone formation was comparable to BMP-2 soaked on collagen but without the ectopic bone formation that limits the clinical efficacy of BMP-2/collagen. In contrast, mechanical loading had no effect on autograft-mediated repair. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.One Sentence SummaryMimicking aspects of the cellular, biochemical, and mechanical environment during early limb development, chondrogenically-primed human mesenchymal stem cell condensations promoted functional healing of critical-sized femoral defects via endochondral ossification, and healing rate and extent was a function of the in vivo mechanical environment.

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1,25(OH)2D3 acts as a bone-forming agent in the hormone-independent senescence-accelerated mouse (SAM-P/6)

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00180.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. E723-E730 ◽

Cited By ~ 32

Author(s):

Gustavo Duque ◽

Michael Macoritto ◽

Natalie Dion ◽

Louis-Georges Ste-Marie ◽

Richard Kremer

Keyword(s):

Bone Formation ◽

Bone Turnover ◽

Bone Marrow Cells ◽

Formation Rate ◽

Mineral Density ◽

Bone Formation Rate ◽

Senile Osteoporosis ◽

Dihydroxyvitamin D ◽

Bone Formation Markers

Recent studies suggest that vitamin D signaling regulates bone formation. However, the overall effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone turnover in vivo is still unclear. In this study, our aim was to examine the effect of 1,25(OH)2D3 on bone turnover in SAM-P/6, a hormone-independent mouse model of senile osteoporosis characterized by a decrease in bone formation. Male and female 4-mo-old SAM-P/6 mice were treated with 1,25(OH)2D3 (18 pmol/24 h) or vehicle for a period of 6 wk, and a group of age- and sex-matched nonosteoporotic animals was used as control. Bone mineral density (BMD) at the lumbar spine increased rapidly by >30 ± 5% ( P < 0.001) in 1,25(OH)2D3-treated SAM-P/6 animals, whereas BMD decreased significantly by 18 ± 2% ( P < 0.01) in vehicle-treated SAM-P/6 animals and remained stable in control animals during the same period. Static and dynamic bone histomorphometry indicated that 1,25(OH)2D3 significantly increased bone volume and other parameters of bone quality as well as subperiosteal bone formation rate compared with vehicle-treated SAM-P/6 mice. However, no effect on trabecular bone formation was observed. This was accompanied by a marked decrease in the number of osteoclasts and eroded surfaces. A significant increase in circulating bone formation markers and a decrease in bone resorption markers was also observed. Finally, bone marrow cells, obtained from 1,25(OH)2D3-treated animals and cultured in the absence of 1,25(OH)2D3, differentiated more intensely into osteoblasts compared with those derived from vehicle-treated mice cultured in the same conditions. Taken together, these findings demonstrate that 1,25(OH)2D3 acts simultaneously on bone formation and resorption to prevent the development of senile osteoporosis.

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The anabolic effect of 17β-oestradiol on the trabecular bone of adult rats is suppressed by indomethacin

Journal of Endocrinology ◽

10.1677/joe.0.1330189 ◽

1992 ◽

Vol 133 (2) ◽

pp. 189-195 ◽

Cited By ~ 7

Author(s):

J. W. M. Chow ◽

J. M. Lean ◽

T. Abe ◽

T. J. Chambers

Keyword(s):

Bone Formation ◽

Trabecular Bone ◽

Bone Growth ◽

Formation Rate ◽

Bone Volume ◽

Female Rats ◽

Mineral Apposition Rate ◽

Adult Rats ◽

Bone Formation Rate

ABSTRACT We have previously demonstrated that administration of oestrogen, at doses sufficient to raise serum concentrations to those seen in late pregnancy, increases trabecular bone formation in the metaphysis of adult rats. To determine whether prostaglandins (PGs), which have been shown to induce osteogenesis in vivo, play a role in the induction of bone formation by oestrogen, 13-week-old female rats were given daily doses of 4 mg 17β-oestradiol (OE2)/kg for 17 days, alone or with indomethacin (1 mg/kg). The rats were also given double fluorochrome labels and at the end of the experiment tibias were subjected to histomorphometric assessment. Treatment with OE2 suppressed longitudinal bone growth and increased uterine wet weight, as expected, and neither response was affected by indomethacin. Oestrogen also induced a threefold increase in trabecular bone formation in the proximal tibial metaphysis, which resulted in a substantial increase in trabecular bone volume. As previously observed, the increase in bone formation was predominantly due to an increase in osteoblast recruitment (as judged by an increase in the percentage of bone surface showing double fluorochrome labels), with only a minor increase in the activity of mature osteoblasts (as judged by the mineral apposition rate). Indomethacin abolished the increase in osteoblastic recruitment, but the activity of mature osteoblastic cells remained high. The bone formation rate and bone volume remained similar to controls. The results suggest that PG production may be necessary for the increased osteoblastic recruitment induced by oestrogen, but not to mediate the effects of oestrogen on the activity of mature osteoblasts. Journal of Endocrinology (1992) 133, 189–195

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CD13 is a Critical Regulator of Cell-cell Fusion in Osteoclastogenesis

10.1101/2020.04.25.061325 ◽

2020 ◽

Author(s):

Mallika Ghosh ◽

Ivo Kalajzic ◽

Hector Leonardo Aguila ◽

Linda H Shapiro

Keyword(s):

Bone Marrow ◽

Bone Formation ◽

Progenitor Cells ◽

Cell Fusion ◽

Formation Rate ◽

Giant Cells ◽

Bone Formation Rate ◽

Cell Cell

AbstractIn vertebrates, bone formation is dynamically controlled by the activity of two specialized cell types: the bone-generating osteoblasts and bone-degrading osteoclasts. Osteoblasts produce the soluble receptor activator of NFκB ligand (RANKL) that binds to its receptor RANK on the surface of osteoclast precursor cells to promote osteoclastogenesis, a process that involves cell-cell fusion and assembly of molecular machinery to ultimately degrade the bone. CD13 is a transmembrane aminopeptidase that is highly expressed in cells of myeloid lineage has been shown to regulate dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. In the present study, we show that CD13-deficient mice display a normal distribution of osteoclast progenitor populations in the bone marrow, but present a low bone density phenotype. Further, the endosteal bone formation rate is similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Loss of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells with remarkably high numbers of nuclei with a concomitant increase in bone resorption activity. Similarly, we also observed increased formation of multinucleated giant cells (MGC) in CD13KO bone marrow progenitor cells stimulated with IL-4 and IL-13, suggesting that CD13 may regulate cell-cell fusion events via a common pathway, independent of RANKL signaling. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP are normally downregulated as fusion progresses in fusion-competent mononucleated progenitor cells, in the absence of CD13 they are uniformly sustained at high levels, even in mature multi-nucleated osteoclasts. Taken together, we conclude that CD13 may regulate cell-cell fusion by controlling expression and localization of key fusion proteins that are critical for both osteoclast and MGC fusion.

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Effect of growth hormone administration and treadmill exercise on serum and skeletal IGF-I in rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1994.266.1.e129 ◽

1994 ◽

Vol 266 (1) ◽

pp. E129-E135 ◽

Cited By ~ 4

Author(s):

J. K. Yeh ◽

J. F. Aloia ◽

M. Chen ◽

N. Ling ◽

H. C. Koo ◽

...

Keyword(s):

Growth Hormone ◽

Growth Factors ◽

Bone Formation ◽

Treadmill Exercise ◽

Formation Rate ◽

Serum Levels ◽

Long Bone ◽

Bone Formation Rate ◽

Hormone Administration ◽

Igf I

Growth factors may be mediators of local and systemic factors that enhance bone formation. This study examined the effect of treadmill exercise and ovine growth hormone administration on levels of insulin-like growth factor I (IGF-I) in serum (ng/ml), long bone, and vertebrae and on bone formation rate. Forty female rats were divided into four groups: control; exercise (17 m/min, 1 h/day); growth hormone (0.05 mg.100 g-1.day-1); growth hormone plus exercise. After 9 wk of study, the serum levels of IGF-I were higher in the intervention groups than in the control group; however, the IGF-I concentration and the periosteal bone formation rate in the long bone were significantly higher only in the exercised rats. The IGF-I concentration and the cancellous bone formation rate in the vertebrae did not differ among the experimental groups. The vertebral and long bone formation rate were correlated with bone concentrations of IGF-I. Serum levels of IGF-I were also correlated with serum osteocalcin and the long bone formation but not with the vertebral bone formation. The association of bone formation with serum and bone IGF-I supports the suggestion that IGF-I is one of the growth factors that regulate bone formation, in particular as a mediator of the response of bone to exercise.

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Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.270.3.e419 ◽

1996 ◽

Vol 270 (3) ◽

pp. E419-E423 ◽

Cited By ~ 12

Author(s):

M. R. Forwood ◽

I. Owan ◽

Y. Takano ◽

C. H. Turner

Keyword(s):

Bone Formation ◽

Formation Rate ◽

Maximal Response ◽

Response Relationship ◽

Bone Formation Rate ◽

Stimulus Response ◽

Threshold Response ◽

Short Period ◽

Single Bout

Based on our quantum concept for mechanically adaptive bone formation, we hypothesized that a single bout of loading would increase bone formation at the endosteal surface in rat tibiae, with a maximal response 4-8 days after loading and a stimulus-response relationship for load magnitude. Bending loads were applied to right tibiae of rats at 31, 43, 53, or 65 N for a single bout of 36 or 360 cycles; bone formation was assessed 1-4, 5-8, or 9-12 days after loading. A single loading episode increased lamellar bone formation rate (BFR) in all groups (P<0.05) and was maximal 5-8 days after loading. A distinct dose-response relationship was not evident among all load magnitudes or for duration, but 65 N was significantly more osteogenic than loads of 31-53 N (P<0.05), consistent with a threshold response to loading. There was also evidence for a significant increase in BFR (P<0.05) and double-labeled surface (P<0.01) within 4 days of loading, suggesting that bone-lining cells were activated directly by the stimulus. Thus subtle changes in BFR may occur by modulating the activity of surface cells, but large modeling drifts and anabolic responses require recruitment and differentiation of osteoprogenitor cells near the bone surface.

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Nck influences preosteoblastic/osteoblastic migration and bone mass

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518253112 ◽

2015 ◽

Vol 112 (50) ◽

pp. 15432-15437 ◽

Cited By ~ 9

Author(s):

Smriti Aryal A.C ◽

Kentaro Miyai ◽

Yayoi Izu ◽

Tadayoshi Hayata ◽

Takuya Notomi ◽

...

Keyword(s):

Cell Migration ◽

Bone Formation ◽

Bone Mass ◽

Formation Rate ◽

Osteoblastic Cell ◽

Bone Formation Rate ◽

Double Deletion ◽

Osteoblastic Lineage ◽

Bone Injury

Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti–IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.

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