Three-dimensional bioprinting of multicell-laden scaffolds containing bone morphogenic protein-4 for promoting M2 macrophage polarization and accelerating bone defect repair in diabetes mellitus

Critical-sized bone defect repair in patients with diabetes mellitus remains a challenge in clinical treatment because of dysfunction of macrophage polarization and the inflammatory microenvironment in the bone defect region.Three-dimensional(3D)bioprinted scaffolds loaded with live cells and bioactive factors can improve cell viability and the inflammatory microenvironment and further accelerating bone repair.Here,we used modified bioinks comprising gelatin,gelatin methacryloyl(GelMA),and 4-arm poly(ethylene glycol)acrylate(PEG)to fabricate 3D bioprinted scaffolds containing BMSCs,RAW264.7 macrophages,and BMP-4-loaded mesoporous silica nanoparticles(MSNs).Addition of MSNs effectively improved the mechanical strength of GelMA/gelatin/PEG scaffolds.Moreover,MSNs sustainably released BMP-4 for long-term effectiveness.In 3D bioprinted scaffolds,BMP-4 promoted the polarization of RAW264.7 to M2 macrophages,which secrete anti-inflammatory factors and thereby reduce the levels of pro-inflammatory factors.BMP-4 released from MSNs and BMP-2 secreted from M2 macrophages collectively stimulated the osteogenic differentiation of BMSCs in the 3D bioprinted scaffolds.Furthermore,in calvarial critical-size defect models of diabetic rats,3D bioprinted scaffolds loaded with MSNs/BMP-4 induced M2 macrophage polarization and improved the inflammatory microenvironment.And 3D bioprinted scaffolds with MSNs/BMP-4,BMSCs,and RAW264.7 cells significantly accelerated bone repair.In conclusion,our results indicated that implanting 3D bioprinted scaffolds containing MSNs/BMP-4,BMSCs,and RAW264.7 cells in bone defects may be an effective method for improving diabetic bone repair,owing to the direct effects of BMP-4 on promoting osteogenesis of BMSCs and regulating M2 type macrophage polarization to improve the inflammatory microenvironment and secrete BMP-2.

Curcumin Inhibits Prostate Cancer Bone Metastasis by Up-Regulating Bone Morphogenic Protein-7 <i>in Vivo</i>

A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-βsignaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP-7). This enhancement of BMP-7 in the context of TGF-β in the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regulated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-β signaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic setting.

Effects of exogenous recombinant human bone morphogenic protein-7 on the corneal epithelial mesenchymal transition and fibrosis

AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibrotic effect via topical application.METHODS：The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells（HECEs）was induced by TGF-β. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid（HA）. EMT markers,fibronectin,E-cadherin,α-smooth muscle actin（α-SMA）,and matrix metaloproteinase-9（MMP-9）,were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7.RESULTS：Treatment with rhBMP-7 attenuated TGF-β-induced EMT in HECEs. It significantly attenuated fibronectin secretion（31.6%; P〈0.05）,the α-SMA protein level（72.2%; P〈0.01）,and MMP-9 expression（23.6%,P〈0.05）in HECEs compared with cells grown in the presence of TGF-β alone. E-cadherin expression was significantly enhanced（289.7%; P〈0.01）in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA~＋ cells by 43.18%（P〈0.05）at a concentration of 2.5 μg/mL and by 47.73%（P〈0.05）at 25 μg/mL,compared with the control group,without disturbing corneal reepithelization.CONCLUSION：rhBMP-7 attenuates TGF-β-induced EMT in vitro,and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-β-related corneal fibrosis with corneal reepithelization disorders.

Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

骨形成蛋白-4对小鼠诱导性多能干细胞牙向分化能力的影响

目的:探讨骨形成蛋白-4(BMP4,bone morphogenetic protein-4)对小鼠诱导性多能干细胞(iPS,induced pluripotent stem cell)牙向分化能力的影响。方法:成釉细胞无血清条件培养液(ASF-CM)中分别加入BMP4(ASF-BMP4)和Noggin(ASF-Noggin),定向诱导小鼠iPS细胞向牙源性细胞转化,观察诱导后细胞形态改变,细胞免疫荧光及RT-PCR检测AMBN、AMGN、CK14、DMP-1、DSPP的表达。结果:免疫荧光染色结果显示:iPS细胞经ASF-BMP4诱导后AMBN、AMGN、CK14、DMP-1、DSPP表达阳性。RT-PCR结果显示:诱导iPS细胞14d后,与ASF-CM组相比,ASF-BMP4组AMBN、DMP-1、DSPP表达显著增高,ASF-Noggin组AMBN、DMP-1、DSPP表达明显降低。结论:BMP4能促进小鼠iPS细胞向成釉细胞谱系和成牙本质细胞谱系分化,而Noggin抑制iPS细胞的分化。

T allele at site 6007 of bone morphogenetic protein-4 gene increases genetic susceptibility to ossification of the posterior longitudinal ligament in male Chinese Han population

Background Several candidate genes of ossification of the posterior longitudinal ligament （OPLL） susceptibility have been identified, but their polymorphisms account for only a small percent of the total variance. Bone morphogenetic protein-4 （BMP4） is a potent ectopic ossification inducing factor. BMP4 protein and mRNA are present in cells from OPLL patients, but not non-OPLL controls. A single nucleotide polymorphism of 6007C〉T（rs17563） of BMP4 has been reported to affect bone density in postmenopausal women. Thus, BMP4 may function in OPLL development. Appropriately, the relationship between BMP4 polymorphisms and OPLL was investigated. Methods A case-control association study investigated the genetic etiology in 179 OPLL patients and 298 non-OPLL controls. Extent of OPLL was analyzed by radiologic examinations. Whether single nucleotide polymorphism （SNP） of -5826G〉A（rs1957860） 5＇ of the transcription start site and 6007C〉T（rs17563） in exon 4 of the BMP4 gene were statistically associated with genetic susceptibility to OPLL in Chinese Han subjects was assessed. Results A significant statistical difference in genotype of 6007C〉T polymorphism between male OPLL patients and male controls was evident, and the frequency of ＂TT＂ genotype in male OPLL patients was significantly higher than in male controls （P=0.039）. The frequency of the ＂T＂ allele was also significantly higher in male OPLL subjects than in male controls （P=0.014, OR=1.57）. A significant difference was also observed between the 6007C〉T polymorphism and the number of ossified cervical vertebrae in OPLL patients, while no statistical difference was apparent between the -5826G〉A polymorphism and OPLL occurrence. Conclusions The T allele in the 6007C〉T polymorphism may be a risk factor for male Han Chinese with ossification of the posterior longitudinal ligament in the cervical spine. Chinese Han male patients with CT and TT 6007C〉T genotypes have a genetic susceptibility to OPLL and more extensive OPLL in the cervical spine.

BMP-4 induced proliferation and oriented differentiation of rat hepatic oval cells into hepatocytes

Objective:To explore the role of bone morphogenetic protein 4(BMP-4) in hepatic progenitor cells(HPCs).Methods:The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line.This hepatocytic cell line could exert various hepatocytc functions including the secretion of albumin and urea.Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist,Noggin,on the proliferation and differentiation of these cells,cellular uptake and excretion of indocyanine green,the periodic acid-schiff(PAS) assay for glycogen storage and the expression of hepatic markers.Results:Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells.Conclusions:This cell source offers a much-needed attractive and expandable source for future investigations of drug screening,stem cell technologies and cellular transplantation,in a society with increasing levels of liver disease and damage.

BMP4在合并糖尿病的冠状动脉旁路移植术患者原位静脉桥病变中的作用

目的观察合并糖尿病的冠状动脉旁路移植术(CABG)患者大隐静脉的形态学变化以及骨形态发生蛋白-4(BMP4)在其管壁中的表达与分布情况,初步探讨BMP4在合并糖尿病患者CABG术后静脉桥病变进展中的潜在作用。方法前瞻性配对纳入2013年1月至2014年12月于我院接受CABG的40例血糖正常患者和40例合并2型糖尿病的患者,并根据糖尿病患者是否需胰岛素治疗分为非胰岛素依赖组(n=24)和胰岛素依赖组(n=16)。应用HE染色测算各组患者静脉桥血管壁的形态学参数,Western blot和免疫组化技术观察BMP4在各组患者静脉管壁中的表达及分布情况。结果糖尿病患者大隐静脉内膜和中膜增生较非糖尿病患者显著。免疫组化显示BMP4主要表达于静脉中膜平滑肌细胞,而BMP4表达量在糖尿病患者大隐静脉管壁内较非糖尿病患者增高(P<0.05)。糖尿病患者静脉桥中BMP4的表达量与其内膜厚度(r=0.655,P<0.01)、内膜面积(r=0.684,P<0.01)、中膜厚度(r=0.642,P<0.01)、中膜面积(r=0.692,P<0.01)均呈明显正相关。结论合并糖尿病的CABG患者大隐静脉内膜和中膜增生程度与管壁内BMP4高表达中度相关,而BMP4可能在此类患者静脉桥加速失效进程中发挥了重要的促进作用。以BMP4为靶点的治疗策略是否有助于改善糖尿病患者CABG术后静脉桥通畅率值得深入研究。

骨形态发生蛋白-4在高糖环境下静脉桥增殖重构进程中的作用研究

目的观察糖尿病大鼠自体静脉桥的形态学变化以及骨形态发生蛋白-4(bone morphogenic protein-4,BMP4)和增殖细胞抗原Ki-67在其管壁中的表达与分布情况,初步探讨BMP4在高糖环境下静脉桥增殖重构进程中的潜在作用。方法选用8周龄的48只雄性Sprague-Dawley大鼠,随机分配为非糖尿病组(即血糖正常组,n=24)及糖尿病组(n=24),采用高脂高糖饮食联合链脲佐菌素的诱导方法构建糖尿病大鼠模型。两组大鼠均建立单侧颈外静脉-颈总动脉桥接模型,每组分别于术前,术后1周、2周和4周获取大鼠静脉桥(n=6)。应用HE染色切片测算大鼠静脉桥血管壁的形态学参数,免疫组化观察Ki-67及BMP4在管壁中的表达及分布情况,逆转录-聚合酶链式反应(RT-PCR)和免疫印迹法(Western blot)检测BMP4基因和蛋白在静脉桥中的表达量。结果在大鼠静脉桥增殖重构进程中,管壁随时间进展不断增厚,而糖尿病大鼠术后内膜和中膜厚度均高于同期的正常大鼠(P<0.05)。免疫组化提示Ki-67表达于大鼠静脉桥管壁平滑肌细胞的胞核,Ki-67的阳性表达随桥血管增殖重构进程逐渐增高,而术后糖尿病大鼠静脉桥内Ki-67的阳性细胞数高于同期的正常大鼠(P<0.05)。免疫组化提示BMP4表达于大鼠静脉桥管壁平滑肌细胞的胞浆,结合RT-PCR及Western blot检测发现,正常大鼠术后静脉桥中仅有少量BMP4的表达,而糖尿病大鼠静脉桥管壁内BMP4的阳性细胞数以及在基因和蛋白水平的表达量均随时间进展逐渐增高,并高于术后同期的正常大鼠(P<0.05)。结论糖尿病大鼠静脉移植术后桥血管增殖性重构改变与管壁内BMP4的高表达密切相关,而BMP4可能在高糖环境下静脉桥加速失效进程中发挥了重要的促进作用。

BMP-4基因／TGF—β1对兔火器性股骨缺损的治疗作用

目的观察BMP-4基因联合TGF—β1对火器性股骨缺损的修复作用。方法用火器性钢珠造成兔股骨中段缺损，伤后2周于骨缺损上、下端和中部注射经体外鉴定的BMP-4基因和TGF—β1。用RT—PCR和Western blot检测BMP-4 mRNA与蛋自在体内的表达，用碱性磷酸酶（ALP）活性和钙含量检测局部组织的成骨能力，用病理学和X线检查成骨过程和质量。结果注射后BMP-4 mRNA和蛋白可在体内表达6周。实验组AIJP活性伤后8周时增加到（13．17±0．51）U／100ml，显著高于对照组的（8．77±0．44）U／100ml，提示实验组的成骨能力增强，钙含量检测与此相符。病理学和X线检查证明实验组成骨过程加快，成骨质量提高。结论BMP-4基因／TGF—β1对火器性骨缺损有明显的促修复作用。

Supercritical fluid-assisted controllable fabrication of open and highly interconnected porous scaffolds for bone tissue engineering

Recently tremendous progress has been evidenced by the advancements in developing innovative three-dimensional(3 D)scaffolds using various techniques for addressing the autogenous grafting of bone. In this work, we demonstrated the fabrication of porous polycaprolactone(PCL) scaffolds for osteogenic differentiation based on supercritical fluid-assisted hybrid processes of phase inversion and foaming. This eco-friendly process resulted in the highly porous biomimetic scaffolds with open and interconnected architectures. Initially, a 2^3 factorial experiment was designed for investigating the relative significance of various processing parameters and achieving better control over the porosity as well as the compressive mechanical properties of the scaffold. Then, single factor experiment was carried out to understand the effects of various processing parameters on the morphology of scaffolds. On the other hand, we encapsulated a growth factor, i.e., bone morphogenic protein-2(BMP-2), as a model protein in these porous scaffolds for evaluating their osteogenic differentiation. In vitro investigations of growth factor loaded PCL scaffolds using bone marrow stromal cells(BMSCs) have shown that these growth factor-encumbered scaffolds were capable of differentiating the cells over the control experiments. Furthermore, the osteogenic differentiation was confirmed by measuring the cell proliferation, and alkaline phosphatase(ALP) activity, which were significantly higher demonstrating the active bone growth. Together, these results have suggested that the fabrication of growth factor-loaded porous scaffolds prepared by the eco-friendly hybrid processing efficiently promoted the osteogenic differentiation and may have a significant potential in bone tissue engineering.