胎盘间充质干细胞治疗小鼠烫伤愈合的相关研究

目的:观察胎盘间充质干细胞对TGF-β1/Smad信号通路的调控作用,探讨胎盘间充质干细胞对烫伤愈合及瘢痕形成的影响。方法:构建小鼠烫伤模型,注射人胎盘间充质干细胞(hPMSCs),荧光显微镜观察小鼠创伤皮肤组织中hPMSCs细胞的存活情况;HE和Masson染色观察小鼠创伤皮肤的变化;Western blot检测观察创伤皮肤TGF-β1、p-Smad3、Smad7、α-SMA、collagen I、Collagen III蛋白表达变化。结果:注射hPMSCs细胞后,小鼠创伤面积逐渐减小,创伤愈合率逐渐增加;hPMSCs细胞分布在小鼠创伤皮肤组织中,存活状况较好。进一步研究发现烫伤模型组皮肤表层细胞受损脱落,真皮层组织疏松,毛囊、皮脂腺等附属器坏死,可见明显的毛细血管扩张,并伴有炎性细胞渗出,同时可见大量的成纤维细胞增生和胶原纤维形成;注射hPMSCs细胞治疗后,病理改变、纤维增生和胶原形成明显减轻;此外,烫伤模型组创伤皮肤组织中TGF-β1、p-Smad3表达明显上调,Smad7蛋白表达明显下调,α-SMA、collagen I、Collagen III表达明显上调。经hPMSCs细胞治疗后,TGF-β1、p-Smad3蛋白表达明显下调,Smad7蛋白表达明显上调,α-SMA、collagen I、Collagen III蛋白表达明显下调。结论:胎盘间充质干细胞可能通过抑制TGF-β1/Smad信号通路,发挥促进烫伤愈合且抑制瘢痕形成的作用。     

Up-regulation of TβRIII facilitates the osteogenesis of supraspinous ligament-derived fibroblasts from patients with ankylosing spondylitis

Spinal supraspinous ligament (SL) osteogenesis is the key risk of ankylosing spondylitis (AS), with an unclear pathogenesis. We previously found that transforming growth factor β1 (TGF-β1), bone morphogenetic proteins (eg BMP2) and type III TGF-β1 receptor (TβRIII) expression were markedly up-regulated in AS-SLs. However, the roles of these closely related molecules in AS are unknown. Here, we showed that BMP2, TGF-β1, TβRIII and S100A4 (a fibroblast marker) were abundant in active osteogenic AS-SL tissues. In vitro, AS-SL fibroblasts (AS-SLFs) showed high BMP2, TGF-β1 and TβRIII expression and auto-osteogenic capacity. We further evaluated the role of TβRIII in the osteogenesis of normal SLFs. BMP2 combined with TGF-β1 induced the osteogenesis of TβRIII-overexpressing SLFs, but the activity was lost in SLFs upon TβRIII knockdown. Moreover, our data suggested that BMP2 combined with TGF-β1 significantly activated both TGF-β1/Smad signalling and BMP2/Smad/RUNX2 signalling to induce osteogenesis of SLFs with TβRIII up-regulation. Furthermore, our multi-strategy molecular interaction analysis approach indicated that TGF-β1 presented BMP2 to TβRIII, sequentially facilitating BMP2 recognition by BMPR1A and promoting the osteogenesis of TβRIII-overexpressing SLFs. Collectively, our results indicate that TGF-β1 combined with BMP2 may participate in the osteogenic differentiation of AS-SLF by acting on up-regulated TβRIII, resulting in excessive activation of both TGF-β1/Smad and BMP2/BMPR1A/Smad/RUNX2 signalling.     

Ⅱ型肺泡上皮细胞间质转分化中的Notch-1/ Twist-1信号通路机制*

目的:通过研究Notch-1/Twist-1信号通路参与Ⅱ型肺泡上皮细胞间质转分化(EMT)作用,为阐明肺纤维化(PF)发病机制提供理论依据。方法:动物实验设对照组和博莱霉素(BLM)组(n=15)。气管注射BLM(7 500 U/kg)诱导肺纤维化大鼠模型,造模后28 d取左肺下叶固定于10%福尔马林中,进行HE、Masson及转化生长因子-β1(TGF-β1)免疫组化染色。体外培养Ⅱ型肺泡上皮细胞(RLE-6TN),细胞实验设对照(Control)组、TGF-β1(5 ng/ml)组、TGF-β1+ Notch-1 siRNA阴性对照组(NC siRNA, 100 pmol/L)和TGF-β1+Notch-1 siRNA干扰组(Notch-1 siRNA, 100 pmol/L),每组设9个复孔。细胞先用NC siRNA或Notch-1 siRNA预处理24 h,再用TGF-β1处理48 h。检测肺组织和(或)II型肺泡上皮细胞内TGF-β1、I型胶原(collagen I)、III型胶原(collagen III)、E-钙粘蛋白(E-Cadherin)、紧密连接蛋白-1(ZO-1)、波形蛋白(Vimentin)、N-钙粘蛋白(N-Cadherin)、Notch-1、Notch-1胞内域(NICD)、Hes-1和Twist-1 mRNA和(或)蛋白表达。结果:动物实验结果显示,与对照组相比,BLM组肺泡萎缩、塌陷并发生融合,肺泡间隔明显增宽,可见大量炎性细胞的浸润。肺间质胶原纤维沉积明显增多,collagen I和collagen III的表达明显增加(P＜0.01);另外,肺BLM组织中E-cadherin和ZO-1表达明显下降而Vimentin和N-cadherin的表达明显增加(P＜0.01);同时,BLM组肺组织TGF-β1、Notch-1、NICD、Hes-1和Twist-1的表达也明显上调(P＜ 0.01)。细胞实验结果显示,与Control相比,TGF-β1组Notch-1、NICD、Hes-1、Twist-1、collagen I和collagen III的表达明显升高,同时E-Cadherin和ZO-1的表达明显降低而Vimentin和N-cadherin的表达明显升高(P＜0.01)。与TGF-β1组相比,Notch-1 siRNA能够明显降低TGF-β1诱导Notch-1、NICD、Hes-1和Twist-1的表达(P＜0.05或P＜ 0.01),同时E-Cadherin和ZO-1的表达明显升高而Vimentin和N-cadherin的表达明显降低(P＜0.05或P＜0.01)。另外Notch-1 siRNA还能够明显降低TGF-β1诱导的collagen I和collagen III的表达(P＜0.05或P＜0.01)。结论:Notch-1/Twist-1信号通路参与了Ⅱ型肺泡上皮细胞EMT,可能参与了肺纤维化的发生发展。     

Platelet-rich plasma promotes the regeneration of cartilage engineered by mesenchymal stem cells and collagen hydrogel via the TGF-β/SMAD signaling pathway

The tissue engineering technique using mesenchymal stem cells (MSCs) and scaffolds is promising. Transforming growth factor-β1 (TGF-β1) is generally accepted as an chondrogenic agent, but immunorejection and unexpected side effects, such as tumorigenesis and heterogeneity, limit its clinical application. Autogenous platelet-rich plasma (PRP), marked by low immunogenicity, easy accessibility, and low-cost, may be favorable for cartilage regeneration. In our study, the effect of PRP on engineered cartilage constructed by MSCs and collagen hydrogel in vitro and in vivo was investigated and compared with TGF-β1. The results showed that PRP promoted cell proliferation and gene and protein expressions of chondrogenic markers via the TGF-β/SMAD signaling pathway. Meanwhile, it suppressed the expression of collagen type I, a marker of fibrocartilage. Furthermore, PRP accelerated cartilage regeneration on defects with engineered cartilage, advantageous over TGF-β1, as evaluated by histological analysis and immunohistochemical staining. Our work demonstrates that autogenous PRP may substitute TGF-β1 as a potent and reliable chondrogenic inducer for therapy of cartilage defect.     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

Activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is needed for the TGFβ-induced chondrogenic and osteogenic differentiation of mesenchymal stem cells

The role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway on the osteogenesis of progenitor and stem cells has received a lot of attention due to conflicting results in the literature. ERK1/2 has been reported to be both activating and inhibitory to the osteogenesis of different cell types under varying culture conditions. This study focused specifically on the role of ERK1/2 on the chondrogenesis and osteogenesis of mesenchymal stem cells (MSC) induced by cytokine exposure. Bone marrow-derived MSC were cultured in three-dimensional fibrin gel scaffolds and stimulated down the chondrogenic and osteogenic programs by addition of TGF-β3 to and osteogenic buffer media. Cells were cultured under control conditions (no cytokine supplementation), treated with TGF-β3 or treated with PD98059 + TGF-β3 for 7 days. RT-PCR results show that addition of TGF-β3 significantly upregulates the phosphorylation of ERK1/2 and induces the cells down the chondrogenic and osteogenic pathways (as demonstrated by the significant upregulation of aggrecan, sox9, collagen types 1 & 2 gene expressions). Inhibition of ERK1/2 phosphorylation with PD98059 led to the abolishment of the upregulation of chondrogenic and osteogenic-specific gene expressions. These results demonstrate that ERK1/2 is needed for the chondrogenic and osteogenic differentiation of MSC as induced by TGF-β3 supplementation.     

Osteoblast-targeted disruption of glucocorticoid signalling does not delay intramembranous bone healing

Objective

Glucocorticoids at pharmacological doses have been shown to interfere with fracture repair. The role of endogenous glucocorticoids in fracture healing is not well understood. We examined whether endogenous glucocorticoids affect bone healing in an in vivo model of cortical defect repair.

Methods

Experiments were performed using a well characterised mouse model in which intracellular glucocorticoid signalling was disrupted in osteoblasts through transgenic overexpression of 11β-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) under the control of a collagen type I promoter (Col2.3-11β-HSD2). Unicortical bone defects (∅0.8 mm) were created in the tibiae of 7-week-old male transgenic mice and their wild-type littermates. Repair was assessed via histomorphometry, immunohistochemistry and microcomputed tomography (micro-CT) analysis at 1-3 weeks after defect creation.

Results

At week 1, micro-CT images of the defect demonstrated formation of mineralized intramembranous bone which increased in volume and density by week 2. At week 3, healing of the defect was nearly complete in all animals. Analysis by histomorphometry and micro-CT revealed that repair of the bony defect was similar in Col2.3-11β-HSD2 transgenic animals and their wild-type littermates at all time-points.

Conclusion

Disrupting endogenous glucocorticoid signalling in mature osteoblasts did not affect intramembranous fracture healing in a tibia defect repair model. It remains to be shown whether glucocorticoid signalling has a role in endochondral fracture healing.     

TGF-β1 induces type I collagen deposition in granulosa cells via the AKT/GSK-3β signaling pathway-mediated MMP1 down-regulation

Type I collagen is the most abundant extracellular matrix (ECM) protein in the mammalian ovary, and comprises two COL1A1 subunits and one COL1A2 subunit. Matrix metalloproteinase 1 (MMP1) is a typical collagenase of type I collagen, that can be detected in ovarian follicles and early corpus luteum. Previous studies demonstrated that MMP1-mediated degradation of type I collagen plays a functional role in regulating corpus luteum formation, and transforming growth factor β1 (TGF-β1) inhibits luteinization and progesterone production in granulosa cells (GCs). Whether TGF-β1 regulates the expression of MMP1, COL1A1, or the deposition of type I collagen during corpus luteum formation remains to be elucidated. This study aimed to investigate the molecular mechanisms through which TGF-β1 regulates MMP1 expression and type I collagen deposition in GCs. Our results show that TGF-β1 upregulates COL1A1 expressions and downregulates MMP1 expression. Inhibition approaches, including pharmacological inhibitors such as p38 inhibitor (SB203580), ERK1/2 inhibitor (U0126), AKT inhibitor (LY294002), and GSK-3β inhibitor (LiCl), as well as knockdown using siRNA specific to these genes, were used. Our results suggest that TGF-β1 decreases MMP1 production via an ALK5-mediated AKT/GSK-3β-dependent signaling pathway, and a decrease in MMP1 levels and an increase in COL1A1 levels synergistically promote type I collagen deposition in GCs. Collectively, these findings provide novel insights into the underlying molecular mechanisms by which TGF-β1 upregulates type I collagen deposition in GCs.     

The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell’s chondrogenic induction.     

Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor β (TGF-β) type I and II receptors, as well as the protein levels of TGF-β1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-β1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.     

Impact of repeated intravenous bone marrow mesenchymal stem cells infusion on myocardial collagen network remodeling in a rat model of doxorubicin-induced dilated cardiomyopathy

Bone marrow mesenchymal stem cells (MSCs) transplantation improved cardiac function and reduced myocardial fibrosis in both ischemic and non-ischemic cardiomyopathies. We evaluated the effects of repeated peripheral vein injection of MSCs on collagen network remodeling and myocardial TGF-β1, AT1, CYP11B2 (aldosterone synthase) gene expressions in a rat model of doxorubicin (DOX)-induced dilated cardiomyopathy (DCM). Thirty-eight out of 53 SD rats survived at 10 weeks post-DOX injection (2.5 mg/kg/week for 6 weeks, i.p.) were divided into DCM blank (without treatment, n = 12), DCM placebo (intravenous tail injection of 0.5 mL serum-free culture medium every other day for ten times, n = 13), and DCM plus MSCs group (intravenous tail injection of 5 × 10⁶ MSCs dissolved in 0.5 mL serum-free culture medium every other day for 10 times, n = 13). Ten untreated rats served as normal controls. At 20 weeks after DOX injection, echocardiography, myocardial collagen content, myocardial expressions of types I and III collagen, TGF-β1, AT1, and CYP11B2 were compared among groups. At 20 weeks post-DOX injection, 8 rats (67 %) survived in DCM blank group, 9 rats (69 %) survived in DCM placebo group while 13 rats (100 %) survived in DCM plus MSCs group. Left ventricular end-diastolic diameter was significantly higher and ejection fraction was significantly lower in DCM blank and DCM placebo groups compared to normal control rats, which were significantly improved in DCM plus MSCs group (all p < 0.05 vs. DCM blank and DCM placebo groups). Moreover, myocardial collagen volume fraction, types I and III collagen, myocardial mRNA expressions of TGF-β1, AT1, CYP11B2, and collagen I/III ratio were all significantly lower in DCM plus MSCs group compared to DCM blank and DCM placebo groups (all p < 0.05). Repeated intravenous MSCs transplantation could improve cardiac function by attenuating myocardial collagen network remodeling possibly through downregulating renin–angiotensin–aldosterone system in DOX-induced DCM rats.     

Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable.     

The antifibrotic effects of TGF-β1 siRNA on hepatic fibrosis in rats

Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-β1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-β1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague–Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-β1 siRNA 0.125 mg/kg treatment group, TGF-β1 siRNA 0.25 mg/kg treatment group and TGF-β1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin–Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-β1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-β1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-β1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-β1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-β1 siRNA negative control group and the TGF-β1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-β1, type I collagen and type III collagen (P < 0.01). Conclusions: Using siRNA to target TGF-β1 can inhibit the expression of TGF-β1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-β1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.     

TGF-β1 diminishes collagen production during long-term cyclic stretching of engineered connective tissue: implication of decreased ERK signaling

Cyclic stretching and growth factors like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.