A novel mechanical loading model for studying the distributions of strain and mechano-growth factor expression

doi:10.1016/j.abb.2011.04.016

Archives of Biochemistry and Biophysics

Volume 511, Issues 1–2, July 2011, Pages 8-13

https://doi.org/10.1016/j.abb.2011.04.016 Get rights and content

Abstract

Mechano-growth factor (MGF), an insulin-like growth factor-I (IGF-I) splice variant, often serves as an important local tissue repair factor in response to the mechanical environment. However, there is no model for exhibiting the MGF expression in a series of strain distribution up to now. In this study, a novel mechanical loading model containing different stresses and strains simultaneously was developed to examine the MGF expression. The strain distributions were predicted by finite element modeling. The MC3T3-E1 cells on a silicone membrane with a central circular hole were exposed to a variable strain environment through stretching. The finite element analysis showed that, when the strain reached the magnitude of 10%, the strain concentration near the circular hole displayed along with the vertical stretch direction, while the minimum strain appeared in the parallel stretch direction. Furthermore, the results showed that MGF expression decreased gradually from high to low strain regions by immunocytochemistry. Meanwhile, the proliferation of osteoblasts increased significantly in the high strain region. In conclusion, this mechanical loading model can present the different distributions of the strain of osteoblasts in vitro. MGF expression and osteoblast proliferation have a high correlation with the levels of strain.

Highlights

► A novel mechanical loading model was developed to examine the MGF expression of the MC3T3-E1 cells in a set of strains distribution. ► The strain distributions of the silicone membrane with a central circular hole were predicted by finite element modeling, not actually measured. ► The strain concentration was near the circular hole in the vertical stretch direction and the minimum strain was in the paralleled direction. ► Mechano-growth factor (MGF) is an alternatively spliced isoform of insulin-like growth factor-I (IGF-I). ► The expression of MGF and PCNA in MC3T3-E1 cells were regulated by mechanical stimulation, depending on the distribution of strain in strain fields.

Introduction

Mechanical stimulation plays crucial roles in modulating the bone growth, remodeling, and repair as well as cellular responses and functions [1], [2], [3], [4], [5]. In recent years it has been demonstrated that a proper stress could promote the bone remodeling and repair [6], [7]. Clinical results confirmed that the distribution of stress surrounding the defective bone was heterogeneous under stress, and the defective bone also appeared stress concentration [8], [9], [10], which may lead to the bone micro-crack and fracture in the regions of strain concentration.

It has been well documented that mechano-growth factor (MGF), an alternative splicing variant of IGF-I [11], is sensitive to mechanical stimuli and/or damage, and it is expressed in bone cells with certain degree of damage. Recently it has been shown to play a critical role in tissue protection and repair [12]. Initially MGF participates in the response to injury in bone cells (or tissues) by autocrine/paracrine [13], suggesting its potentially positive role in bone remodeling. Osteoblasts in bone are the sensitive cells responsible for perceiving the stress or strain signals during the mechanical signal transduction. The osteoblasts expressing MGF in stretching environment has been documented [13], [14], [15]. However, the distribution of MGF expression related to strain concentration remains essentially unknown.

In present, we focused on quantifying the relation between the deformations of substrate with a central circular hole and the distribution of MGF expression, as well as osteoblast proliferation under mechanical loading.

Section snippets

Preparation of the uniaxial mechanical loading model

The uniaxial mechanical loading model was a rectangular silicone membrane with a central circular hole. The silicone membrane was made by Sylgard 184 Silicone Elastomer (Dow Corning, Midland-Michigan, USA). The hole in the center of the membrane was made by a punch utensil. The edge of the hole was considered smooth. The physical and geometric properties of the silicone membrane were shown in Table 1.

The finite element analysis

In this study, to simplify the question, only the mechanical simulation with static loading was

The finite element analysis of the uniaxial mechanical loading model

When the strain reached the magnitude of 10%, the strain concentration near the circular hole displayed along with the vertical stretch direction (Fig. 2A), and the stress gradually decreased from the region of near the hole to the edge of the membrane beneath the central circular hole (Fig. 2C). The strain varied from 23.1% near the hole to 9.6% at the edge of the membrane (Fig. 2B). While the minimum strain appeared in the parallel stretch direction (Fig. 2A), and the stress gradually

Discussion

The previous studies have detected the MGF expression after constant magnitude on osteoblasts in vitro [13], [14], [17], [18]. However, bone cells are exposed to a heterogeneous strain environment in vivo. To better understand how bone cells adapt to the complicated environment through regulating the expression of MGF, a new mechanical loading model with a series of strain distribution was designed in this study. The results demonstrated that the distribution of strain in the different regions

Conclusion

In conclusion, the silicone membrane with a central circular hole with cyclic uniaxial stretching model could efficiently simulate the loading situation of bone in different mechanical environments. The expression of MGF and PCNA in MC3T3-E1 cells were regulated by mechanical stimulation, depending on the distribution of strain in strain fields. The expression of MGF gradually diminished from high strain regions to low strain regions. The distribution of PCNA expression was similar to the

Acknowledgments

This work was supported by Grants from the National Natural Science Foundation of China (1032012, 30870609), Natural Science Foundation of CQ CSTC (2009BB4382, 2010BB5225), Science and Technology Program of CQ CSTC (2009AB517), Foundation of Chongqing Municipal Education Commission (KJ091415) and the Fundamental Research Funds for the Central Universities (CDJXS11230040).

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Numerous growth factors are involved in the regulation of tissue repair. Mechano growth factor (MGF) is a splice variant of the insulin-like growth factor; it mainly acts in an autocrine or paracrine manner [26,27] and is upregulated in osteoblasts [28], cardiomyocytes [29], and neurons [30], allowing them to withstand mechanical stimulation or hypoxia-ischemia, thereby protecting the cells from damage or facilitating tissue repair. The N-terminal E domain of MGF (MGF E peptide) has a similar function as MGF in promoting tissue repair or maintaining cell physiology under disadvantageous environments.
Osteoarthritis (OA) is characterized by pain and declining gait function associated with degeneration of cartilage. A severe hypoxic environment occurs due to tissue injury in the joint cavity and may aggravate the development of OA. In this study, the effects of severe hypoxia and treatment with mechano growth factor (MGF) E peptide on metabolism of the extracellular matrix (ECM) during the progression of OA were determined. The results showed that cell viability, cell proliferation, and type II collagen expression in chondrocytes were significantly inhibited by cobalt chloride (CoCl₂)-simulated severe hypoxia, whereas cell apoptosis and expression levels of hypoxia inducible factor 1 alpha, type I collagen, and matrix metalloproteinases 1/13 were clearly induced. Pretreatment with MGF E peptide reduced the abovementioned adverse effects induced by CoCl₂-simulated severe hypoxia in chondrocytes. Pretreatment also upregulated the proliferation of chondrocytes under severe hypoxia through the PI3K-Akt and MEK-ERK1/2 signaling pathways. In a rat model of monosodium iodoacetate (MIA)-induced OA. MIA treatment induced tissue necrosis and cartilage degeneration, and histological score was significantly decreased. The levels of type II collagen and aggrecan were reduced after MIA treatment for 4 or 6 weeks, and abnormal distribution of ECM occurred in the inner epicondyle after 6 weeks. MGF E peptide also reduced the progression of MIA-induced OA by retarding cartilage degeneration, upregulating type II collagen synthesis, and improving ECM distribution after 4 or 6 weeks. Our findings suggest that MGF attenuates the progression of OA, and thus may be applied for the treatment of OA in the clinic.
Mechano-growth factor protects against mechanical overload induced damage and promotes migration of growth plate chondrocytes through RhoA/YAP pathway
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MGF is an autocrine and endocrine growth factor which is expressed in many tissues [7]. It was reported by previous studies that MGF could regulate proliferation, differentiation and migration in a variety of tissue types and exerted protective functions against different, often harmful stimuli [30]. However, molecular mechanisms by which MGF exerts its function were not fully understood.
Epiphyseal growth plate is highly dynamic tissue which is controlled by a variety of endocrine, paracrine hormones, and by complex local signaling loops and mechanical loading. Mechano growth factor (MGF), the splice variant of the IGF-I gene, has been discovered to play important roles in tissue growth and repair. However, the effect of MGF on the growth plate remains unclear. In the present study, we found that MGF mRNA expression of growth plate chondrocytes was upregulated in response to mechanical stimuli. Treatment of MGF had no effect on growth plate chondrocytes proliferation and differentiation. But it could inhibit growth plate chondrocytes apoptosis and inflammation under mechanical overload. Moreover, both wound healing and transwell assay indicated that MGF could significantly enhance growth plate chondrocytes migration which was accompanied with YAP activation and nucleus translocation. Knockdown of YAP with YAP siRNA suppressed migration induced by MGF, indicating the essential role of YAP in MGF promoting growth plate chondrocytes migration. Furthermore, MGF promoted YAP activation through RhoA GTPase mediated cytoskeleton reorganization, RhoA inhibition using C3 toxin abrogated MGF induced YAP activation. Importantly, we found that MGF promoted focal adhesion(FA) formation and knockdown of YAP with YAP siRNA partially suppressed the activation of FA kinase, implying that YAP is associated with FA formation. In conclusion, MGF is an autocrine growth factor which is regulated by mechanical stimuli. MGF could not only protect growth plate chondrocytes against damage by mechanical overload, but also promote migration through activation of RhoA/YAP signaling axis. Most importantly, our findings indicate that MGF promote cell migration through YAP mediated FA formation to determine the FA-cytoskeleton remodeling.
MGF E peptide pretreatment improves the proliferation and osteogenic differentiation of BMSCs via MEK-ERK1/2 and PI3K-Akt pathway under severe hypoxia
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Numbers of studies confirm that MGF E peptide (abbreviated as MGF in the following) has similar functions with MGF in promoting cell proliferation, activating satellite cells, maintaining nerve survival, and regulating tissue healing [16]. MGF is always up-regulated in some kinds of cells or tissues withstanding high-intensity mechanical stimulation or oxygen tension [17–19]. It was reported that MGF could protect myocytes and neurons from hypoxia [20,21].
Severe hypoxia always inhibits the cell proliferation, osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and hinders bone defect repair. Herein we explored the effects of mechano-growth factor (MGF) E peptide on the proliferation and osteogenic differentiation of BMSCs under severe hypoxia.
CoCl₂ was utilized to simulate severe hypoxia. MTS was used to detect cell viability. Cell proliferation was verified through flow cytometry and EdU assay. Osteogenic differentiation of BMSCs and osteoblast-specific genes were detected through alkaline phosphatase (ALP) and Alizarin Red S staining, and quantitative real-time PCR, respectively. Hypoxia-inducible factor 1α (HIF-1α), p-ERK1/2 and p-Akt expression levels were detected through western blotting and immunofluorescence.
Severe hypoxia induced HIF-1α accumulation and transferring into the nucleus, and reduced cell proliferation and osteogenic differentiation of BMSCs. The expression levels of osteoblast-specific genes were markedly decreased after differentiation culture for 0, 7 or 14 days. Fortunately, MGF E peptide inhibited HIF-1α expression and transferring into the nucleus. Cell proliferation and osteogenic differentiation of BMSCs could be recovered by MGF E peptide pretreatment. MEK-ERK1/2 and PI3K-Akt signaling pathway were confirmed to be involved in MGF E peptide regulating the abovementioned indexes of BMSCs. What's more, short-time treatment with MGF E peptide alone promoted the osteogenic differentiation of BMSCs as well.
Our study provides new evidence for the role of MGF E peptide in regulating proliferation and osteogenic differentiation of BMSCs under severe hypoxia, which may potentially have therapeutic implication for bone defect repair.
Microencapsulation of mechano growth factor e peptide for sustained delivery and bioactivity maintenance
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Mechano growth factor (MGF) is a novel bioactive molecule, which is originated from alternative splicing of insulin-like growth factor-I (IGF-I) gene. It is prominently expressed in over-stretched mechanically responsive tissues, such as skeletal muscle and bone (Goldspink, 2005; Peng et al., 2011). MGF has C-terminal E-peptide with 24 amino acids, MGF-Ct24E, which is different from other IGF-I isoforms.
Mechano growth factor (MGF) and its C-terminal E-peptide with 24 amino acids, MGF-Ct24E, have superiority in resolving the delayed or failed bone repair derived from shortness of suitable biomechanical stimulation. The chitosan/tripolyphosphate microspheres encapsulated with MGF-Ct24E (CS/TPP/MGF-Ct24E) are prepared using emulsion-ionic cross-linking method in order to achieve the sustained release and preserve the bioactivity of MGF-Ct24E. The microspheres are micron-sized and spherical in shape with smooth surface morphology. The TPP component disintegrates in advance of CS matrix and the MGF-Ct24E maintains sustained delivery during in vitro hydrolytic degradation. With the disappearance of TPP, the total weight loss of CS/TPP/MGF-Ct24E is 32% and the release amount of MGF-Ct24E reaches 84.6% after degrading for 2 weeks. In vitro bioactivity assays reveal that the MGF-Ct24E can accelerate MC3T3-E1 cells proliferation and delay their differentiation as well. The encapsulated MGF-Ct24E shows long-term effects after being loaded in the CS/TPP microspheres and the cells exhibit excellent morphology on the surface of microspheres. The continuous delivery of MGF-Ct24E provides a new perspective on resolving the unsatisfactory bone reconstruction associated with microgravity and stress shielding.
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A novel mechanical loading model for studying the distributions of strain and mechano-growth factor expression

Abstract

Highlights

Introduction

Section snippets

Preparation of the uniaxial mechanical loading model

The finite element analysis

The finite element analysis of the uniaxial mechanical loading model

Discussion

Conclusion

Acknowledgments

J. Biomech.

Growth Horm. IGF Res.

Arch. Oral Biol.

Biochem. Biophys. Res. Commun.

J. Mol. Cell Cardiol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Biomech.

J. Biomech.

J. Biomech.

J. Biomech.

J. Biomech.

Calcif. Tissue Int.

J. Bone Miner Metab.

J. Bone Miner Res.

Instr. Course Lect.

Calcif. Tissue Int.