Cyclic tensile stretch modulates osteogenic differentiation of adipose-derived stem cells via the BMP-2 pathway

Introduction

Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals.

Material and methods

We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µɛ) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µɛ). Then osteogenic related genes were analysed by real-time PCR.

Results

The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h’ duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP.

Conclusions

We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.     

Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究

目的　利用重组Runx2基因的慢病毒感染大鼠骨髓间充质干细胞（BMSCs）,使其Runx2基因的表达水平提高,观察BMSCs成骨相关基因的表达情况,研究Runx2基因促进BMSCs的成骨分化情况。 方法　PCR扩增Runx2基因,并连接到慢病毒表达载体质粒Pez-lv201,与包装质粒共转染293T细胞进行包装,测得慢病毒液滴度。取4周龄SD大鼠胫骨,分离、培养BMSCs,流式细胞仪鉴定。将Runx2重组慢病毒感染BMSCs。显微镜下观察细胞形态变化;RT-PCR分析BMSCs成骨基因的表达情况。 结果　Runx2基因重组慢病毒表达载体质粒酶切和测序鉴定正确。测得慢病毒液滴度为1.6×109 TU/ml。流式细胞仪检测表面抗原CD90和CD105,表达率为99.8%、99.3%。重组慢病毒感染后实验组细胞形态呈成骨样细胞改变;对照组未见明显变化。实验组Runx2、OCN、osteonectin、ALP、BMP-2、OPN基因的表达水平随时间推移而增高;对照组上述基因均无明显表达。 结论　利用Runx2重组慢病毒感染BMSCs,使其高表达Runx2基因,可以使OCN、osteonectin、ALP、BMP-2、OPN基因表达增强,说明Runx2基因可以促进BMSCs向成骨方向分化。     

低氧条件下载二甲基草酰甘氨酸电纺纤维促血管化及成骨性能研究

目的 通过聚L-丙交酯-己内酯电纺纤维（PLCL）负载二甲基草酰甘氨酸（DMOG）,研究其在低氧和常氧成骨诱导过程中对大鼠骨髓间充质干细胞（BMSC）的血管化和体外成骨分化的作用。方法 本研究经静电纺丝技术制备PLCL电纺纤维（P）和负载DMOG的PLCL电纺纤维（PD）,通过扫描电镜观察电纺纤维形貌;通过细胞骨架染色观察BMSC在不同电纺纤维上的黏附和生长状态;通过碱性磷酸酶和茜素红染色检测在不同电纺纤维上的BMSC经常氧和低氧成骨诱导7 d后的碱性磷酸酶表达和14 d时的钙沉积情况;通过RT-PCR检测在不同电纺纤维上的BMSC经常氧和低氧成骨诱导7 d和14 d时的成骨相关基因（ALP、Runx2、Col1和OCN）和促血管化相关基因（VEGF）的表达情况。结果 扫描电镜结果表明,P和PD具有纤维状形态并呈现为多孔结构。细胞实验表明,BMSC可在P和PD表面黏附生长,且低氧条件下在PD上表现出更好的形态。与常氧条件相比,在低氧条件下,P和PD在7 d时的碱性磷酸酶表达减少。但低氧条件成骨诱导14 d时PD仍能促进钙的沉积。在常氧条件下,电纺纤维P可上调ALP、Runx2、Col1、OCN和VEGF的表达,但低氧条件下其对上述基因的上调作用不明显。而电纺纤维PD在常氧和低氧条件下均可促进ALP、Runx2、Col1、OCN和VEGF的表达。结论 本研究制备的负载DMOG的PLCL电纺纤维在低氧条件下具有良好的体外促血管化和促成骨分化性能,预期可作为一种较好的成骨修复材料。     

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats

AimTo compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects.MethodThe endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting.ResultEndogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower.ConclusionThe superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.     

Expression of SP7/osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen huoxue decoction北大核心

BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Ectopic and in situ bone formation of adipose tissue-derived stromal cells in biphasic calcium phosphate nanocomposite

Adipose-derived stromal cells (ASCs) have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. A novel biodegradable biphasic calcium phosphate nanocomposite (NanoBCP) comprising beta-tricalcium phosphate matrix and hydroxyl apatite nanofibers is favorable for bone tissue engineering. In this study, ASCs were harvested from Sprague-Dawley (SD) rats and induced to osteogenesis before seeded into porous NanoBCP scaffold. To determine ectopic in vivo osteogenic differentiation, these constructs were implanted in nude mice subcutaneously. Meanwhile, the ability of engineered constructs to stimulate in situ bone repair was assessed in rat critical-size cranial defects. The defects were filled with NanoBCP containing osteogenic ASCs in experimental group; with cell-free NanoBCP in negative controls; and with nothing in blank controls. The retrieved specimens were analyzed with morphological, histological, and molecular methods. Histological analysis of the retrieved specimens from nude mice in experimental group showed obvious ectopic bone formation. There were positive expression of osteopontin (OPN) and osteocalcin (OCN) at RNA and protein level. As for the cranial defects, there was complete repair in experimental group, but only partial repair in negative controls. The radiographs, H&E staining, and Masson's trichrome method showed better bone regeneration at experimental sites. Combining osteogenic ASCs with NanoBCP can lead to formation of ectopic new bone. Furthermore, the approach can also stimulate bone regeneration and repair for the large size bone defects.     

Solution-mediated effect of bioactive glass in poly (lactic-co-glycolic acid)-bioactive glass composites on osteogenesis of marrow stromal cells

A previous study demonstrated that the incorporation of bioactive glass (BG) into poly (lactic-co-glycolic acid) (PLGA) can promote the osteoblastic differentiation of marrow stromal cells (MSCs) on PLGA by promoting the formation of a calcium-phosphate-rich layer on its surface. To further understand the mechanisms underlying the osteogenic effect of PLGA-BG composite scaffolds, whether solution-mediated factors derived from composite scaffolds/hybrids can promote osteogenesis of marrow stromal cells was tested. The dissolution product from PLGA-30%BG scaffold stimulated osteogenesis of MSCs, as was confirmed by increased mRNA expression of osteoblastic markers such as osteocalcin (OCN), alkaline phosphatase (ALP), and bone sialoprotein (BSP). The three-dimensional structure of the scaffolds may contribute to the production of cell-derived factors that promoted distant MSC differentiation. Thus PLGA-BG composites demonstrate significant potential as a bone-replacement material.     

Cyclic tension promotes osteogenic differentiation in human periodontal ligament stem cells

Orthodontic forces result in alveolar bone resorption and formation predominantly on the pressure and tension sides of the tooth roots, respectively. Human periodontal ligament stem cells (PDLSCs) have demonstrated the capacity to differentiate into osteoblasts, and they play important roles in maintaining homeostasis and regenerating periodontal tissues. However, little is known about how PDLSCs contribute to osteoblastogenesis during orthodontic tooth movement on the tension side. In this study, we applied a 12% cyclic tension force to PDLSCs cultured in osteoinductive medium. The osteogenic markers Runx2, ALP, and OCN were detected at the mRNA and protein levels at different time points using real-time PCR and western blot analyses. We discovered that the mRNA and protein levels of Runx2, ALP and OCN were significantly up-regulated after 6, 12 and 24 hours of mechanical loading on PDLSCs compared to levels in unstimulated PDLSCs (P < 0.05). This study demonstrates, for the first time, the effects of mechanical tensile strain on the osteogenic differentiation of PDLSCs, as examined with a Flexcell FX-4000T Tension Plus System. Our findings suggested that cyclic tension could promote the osteogenic differentiation of PDLSCs. Furthermore, the effects of orthodontic force on alveolar bone remodeling might be achieved by PDLSCs.     

Osteogenic differentiation of osteoblasts induced by calcium silicate and calcium silicate/β-tricalcium phosphate composite bioceramics

In this study, calcium silicate (CS) and CS/β-tricalcium phosphate (CS/β-TCP) composites were investigated on their mechanism of osteogenic proliferation and differentiation through regulating osteogenic-related gene and proteins. Osteoblast-like cells were cultured in the extracts of these CS-based bioceramics and pure β-TCP, respectively. The main ionic content in extracts was analyzed by inductively coupled plasma-atomic emission spectroscopy. The cell viability, mineralization, and differentiation were evaluated by MTT assay, Alizarin Red-S staining and alkaline phosphatase (ALP) activity assay. The expressions of BMP-2, transforming growth factor-β (TGF-β), Runx2, ALP, and osteocalcin (OCN) at both gene and protein level were detected by real-time polymerase chain reaction analysis and Western blot. The result showed that the extracts of CS-based bioceramics promoted cells proliferation, differentiation, and mineralization when compared with pure β-TCP. Accordingly, pure CS and CS/β-TCP composites stimulated osteoblast-like cells to express BMP-2/TGF-β gene and proteins, and further regulate the expression of Runx2 gene and protein, and ultimately affect the ALP activity and OCN deposition. This study suggested that the CS-based bioceramics could not only promote the expression of osteogenic-related genes but also enhance the genes to encode the corresponding proteins, which could finally control osteoblast-like cells proliferation and differentiation.     

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.     

Bone tissue engineering using human adipose-derived stem cells and honeycomb collagen scaffold

Human adipose-derived stem cells (ASCs) have the capacity to regenerate and the potential to differentiate into multiple lineages of mesenchymal cells. The aim of this study was to investigate the possibility of using honeycomb collagen scaffold to culture ASCs in bone tissue engineering. The osteogenic capacity of ASCs in vitro, was confirmed by histology and measuring the expression of cbfa-1. After that, ASCs were cultured for up to 14 days in the honeycomb scaffold to allow a high density, three-dimensional culture. Scanning electron microscopy data showed that the scaffold was filled with the grown ASCs, and calcification, stained black with von Kossa, was confirmed. Furthermore, The ASC-loaded honeycomb collagen scaffolds cultured for 14 days were subcutaneously transplanted into nude mice, and excised after 8 weeks. Bone formation in vivo was examined using HE stain, von Kossa stain, and osteocalcin immunostain. Those histological views showed significant positive stains in the samples of osteogenic medium in the three types of stain. These results suggest that this carrier is a suitable scaffold for ASCs and will be useful as a three-dimensional bone tissue engineering scaffold in vitro and in vivo.     

Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds

Boron is one of the trace elements in the human body which plays an important role in bone growth. Porous mesopore bioactive glass (MBG) scaffolds are proposed as potential bone regeneration materials due to their excellent bioactivity and drug-delivery ability. The aims of the present study were to develop boron-containing MBG (B-MBG) scaffolds by sol-gel method and to evaluate the effect of boron on the physiochemistry of B-MBG scaffolds and the response of osteoblasts to these scaffolds. Furthermore, the effect of dexamethasone (DEX) delivery in B-MBG scaffold system was investigated on the proliferation, differentiation and bone-related gene expression of osteoblasts. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of B-MBG scaffolds have been characterized. The effect of boron contents and large-pore porosity on the loading and release of DEX in B-MBG scaffolds were also investigated. The results have shown that the incorporation of boron into MBG scaffolds slightly decreases the specific surface area and pore volume, but maintains well-ordered mesopore structure and high surface area and nano-pore volume compared to non-mesopore bioactive glass. Boron contents in MBG scaffolds did not influence the nano-pore size distribution or the loading and release of DEX. B-MBG scaffolds have the ability to maintain a sustained release of DEX in a long-term span. Incorporating boron into MBG glass scaffolds led to a controllable release of boron ions and significantly improved the proliferation and bone-related gene expression (Col I and Runx2) of osteoblasts. Furthermore, the sustained release of DEX from B-MBG scaffolds significantly enhanced alkaline phosphatase (ALP) activity and gene expressions (Col I, Runx2, ALP and BSP) of osteoblasts. These results suggest that boron plays an important role in enhancing osteoblast proliferation in B-MBG scaffold system and DEX-loaded B-MBG scaffolds show great potential as a release system to enhance osteogenic property for bone tissue engineering application.     

Restoration of critical-size defects in the rabbit mandible using porous nanohydroxyapatite-polyamide scaffolds

Composite nanohydroxyapatite/polyamide (n-HA/PA) biomaterials have been indicated for bone defect reconstruction, where PA is added to enhance the toughness of n-HA. However, a comprehensive understanding of the biological performance of this implant material remains to be determined. In this study, the biological activity of n-HA/PA biomaterials was characterized in vitro by assessing the growth of bone marrow stromal cells (BMSCs), and in an in vivo rabbit model. To evaluate the n-HA/PA performance under different osteogenic conditions in vivo, implants were inserted to critical-size bone defects in the angle and body of the rabbit mandible. To determine the necessity of ectogenic BMSC-n-HA/PA hybrids at different implantation sites, both raw n-HA/PA materials and BMSC-seeded n-HA/PA hybrids were implanted. Bone formation was detected by radiology and histological studies. The results showed that n-HA/PA composites had great bioactivity, demonstrating significant BMSC proliferation, active alkaline phosphatase secretion, and stimulating the expression of osteogenic proteins (bone morphogenetic protein 2 [BMP2], osteoprotegerin [OPG], osteopontin [OPN], collagen type I [Col I], and osteocalcin [OCN]), in comparison to the control (polyethylene). At marrow-rich implantation sites (mandibular body), the amount of new bone formation was significant, but was not enhanced by the presence of BMSCs in the BMSC-n-HA/PA hybrids. However, the BMSC-n-HA/PA hybrids were essential for promoting bone formation in marrow-poor sites (mandibular angle). In conclusion, n-HA/PA biomaterials, which offer the advantage of enhanced mechanical performance over n-HA, exhibit significant bioactivity, including the capacity for bone regeneration at marrow-poor sites when implanted in combination with BMSCs.