Bone matrix insulin-like growth factor (IGF)-I, IGF-II and transforming growth factor (TGF)-beta1 levels in men and postmenopausal women with osteoporosis: lack of association with circulating growth factors and bone mineral density

Previous clinical studies have suggested a positive correlation between serum insulin-like growth factor components and bone mass in both men and women with or without osteoporosis. The aim of the present study was to analyze the relationship between the skeletal levels of insulin-like growth factors and transforming growth factor-b1 and bone mineral density in a group of men and postmenopausal women in whom osteoporosis was diagnosed previously. Bone matrix extraction was achieved by passive dialysis against tetrasodium EDTA-guanidine-HCL. IGF's were quantified by radioimmunoassay. TGF-b1 was assessed by a specific enzyme-linked immunoassay. No correlation between BMD and the concentration of IGF-I, IGF-II and TGF-b1 in bone matrix was detected in either men or postmenopausal women with osteoporosis. In addition, circulating growth factors levels failed to be associated with the concentration of IGF-I, IGF-II and TGF-b1 in the skeleton. Thus, our study provides no evidence for a major role of bone matrix IGF's or TGF-b1 as determinants of bone mass in men or postmenopausal women with osteoporosis.     

Expression of dentin matrix protein 1 (DMP1) during fracture healing

Dentin matrix protein 1 (DMP1) is one of the acidic phosphorylated extracellular matrix proteins called the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family. Recent studies showed that DMP1 is expressed in the mineralized tissues and suggested that DMP1 is involved in the mineralization. We investigated the precise localization of DMP1 messenger RNA (mRNA) and protein during fracture healing. In situ hybridization demonstrated that DMP1 mRNA was strongly expressed in preosteocytes and osteocytes in the bony callus during intramembranous and endochondral ossification while DMP1 mRNA was not detected in osteoblasts and chondrocytes. During endochondral ossification, however, a low number of DMP1-expressing cells were identified in the cluster of hypertrophic chondrocytes. However, these DMP1-expressing cells were not hypertrophic and were likely to be osteoblast-lineage cells, which were embedded in the matrix of bone or cartilage, because type I collagen-expressing cells and invasion of capillary vessels were observed in the same area. Northern blot, in situ hybridization, and immunohistochemical analyses showed that DMP1 mRNA and protein expressions were increased until day 14 postfracture, when bony callus was formed, and then declined to a lower level during remodeling of the bony callus. Therefore, DMP1 is likely to play an important role in the mineralization of the bony callus.     

Effect of heparin on production of transforming growth factor (TGF)-beta1 and TGF-beta1 mRNA expression by human normal skin and hyperplastic scar fibroblasts

Heparin affects both dermal fibroblast proliferation and collagen and may mediate these effects by altering the levels of transforming growth factor-beta1 (TGF-beta1) production and TGF-beta1 mRNA expression as a wound healing modulator. The purpose of this study is to probe the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts. This research investigates the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts with exposure to 0 microg/mL, 100 microg/mL, 300 microg/mL, or 600 microg/mL heparin for 24, 48, 72, or 96 hours in a serum-free in vitro model. Levels of TGF-beta1 in the supernatants and TGF-beta1 mRNA expression of fibroblasts were determined by enzyme-linked immunosorbent assay (ELISA) and real time RT-PCR, respectively. Heparin (300 microg/mL and 600 microg/mL) stimulated TGF-beta1 production by normal skin (26% to 83%) and hyperplastic scar fibroblasts (63% to 85%), with statistical significance (P < 0.05) at various time points. Heparin (300 microg/mL and 600 microg/mL) also stimulated TGF-beta1 mRNA expression by normal skin (12% to 53%) and hyperplastic scar fibroblasts (33% to 52%), with statistical significance (P < 0.05) at various time points. These effects of heparin on normal skin and hyperplastic scar fibroblasts may have implications for hyperplastic scar formation and wound healing in vivo.     

鞘注神经生长因子促进大鼠胫骨骨折愈合的实验研究

目的 研究鞘注神经生长因子(NGF)对大鼠胫骨骨折愈合的影响,以及相应脊髓背根神经节(DRG)与胫骨骨痂中的降钙素基因相关肽、P物质的表达变化. 方法 成年雄性SD大鼠48只随机分为两组.实验组予NGF 1μg/d连续鞘注14 d,对照组予等量生理盐水.术后7、14、21、28 d分批处死动物,行骨痂X线评分、骨痂/骨干比值测量.免疫组织化学法测定相应节段DRG与胫骨骨痂中的CGRP、SP表达.结果 术后21 d,NGF组X线评分低于对照组,21 d、28 d骨痂/骨干比值低于对照组.HE染色显示各时期NGF组软骨内成骨过程增强,骨痂成熟度高于对照组,骨痂改建过程提前且更为完全.CGRP、SP在DRG及胫骨骨痂中表达的平均光密度(OD)值高于同期对照组,差异有统计学意义(P＜0.05).相关性分析显示DRG神经肽表达OD值与骨痂OD值明显正相关. 结论 鞘注神经生长因子可促进DRG与骨折骨痂中的神经肽表达,促进成骨细胞增殖与软骨内骨化,减小骨痂体积并加速骨折的愈合及塑形改建过程.推测神经生长因子与DRG神经肽参与了中枢神经调控外周成骨活动的过程.     

Expression of epidermal growth factor and epidermal growth factor receptor genes in healing colonic anastomoses in rats.

This study uses complementary DNA probes to find out if epidermal growth factor (EGF) and EGF receptor genes are activated in healing colonic anastomoses in rats. 35 rats had a colocolonic anastomosis which was removed for study 1, 2, 3, 5, 7, or 14 days after operation. Six animals without surgery served as controls. The specimens were examined by northern blotting, in situ hybridisation, and conventional microscopy. Microscopic healing progressed normally. The expression of the EGF gene was minimal and surgery did not activate it. However, surgical trauma did increase the expression of the EGF receptor gene by 2.2 times when compared to day one. In situ hybridisations localised a strong EGF receptor expression in mucosal epithelial cells in all specimens, and a moderate reaction in fibroblasts in the repair tissue in the anastomotic line. The enhanced EGF receptor gene expression suggests that anastomotic healing is associated with the presence of EGF or an EGF-like substance and its activity increases during the first postoperative week.     

血管紧张素Ⅱ受体拮抗剂对肝硬化大鼠TGFβ1基因表达影响的实验研究

目的研究血管紧张素Ⅱ(AngiotensinⅡ)Ⅰ型受体(AT1)拮抗剂Losartan对肝硬化大鼠TGF β1(transforming growth factor β1,转化生长因子β1)基因表达的影响.方法 41只雄性SD大鼠分为4组对照组(10只)、模型组(11只)、及Losartan治疗组(20只)[早期(10)、中期(10)],除对照组外所有大鼠均给予50% CCL4灌胃,3*!mg*kg-1*5d-1,共9周.治疗组早期组同时给予血管紧张素受体拮抗剂Losartan灌胃,中期组于造模中期(5周)开始给药,用量10*!mg*kg-1*d-1至处死前.实验结束后,处死取肝脏液氮冻存及福尔马林固定标本,分别行免疫组织化学及RT-PCR检测,TGF β1蛋白及基因表达情况.结果 与正常组相比,模型组TGF β1表达明显增强,由0.93±0.05升高到1.94±0.13(P<0.01),而两治疗组与模型组相比,losartan明显降低了TGF β1 mRNA的表达,分别降至1.17±0.16、1.02±0.11(P<0.05),而早、中期两组差异无显著意义(P>0.05).结论 Losartan对肝硬化时TGF β1表达有明显抑制作用.     

Evidence for a role of transforming growth factor (TGF)-beta1 in the induction of postglomerular albuminuria in diabetic nephropathy: amelioration by soluble TGF-beta type II receptor

Transforming growth factor-beta (TGF-beta) has previously been implicated in the progression of diabetic nephropathy, including the onset of fibrosis and albuminuria. Here we report for the first time the use of a high-affinity TGF-beta1 binding molecule, the soluble human TGF-beta type II receptor (sTbetaRII.Fc), in the treatment of diabetic nephropathy in 12-week streptozotocin-induced diabetic Sprague-Dawley rats. In vitro studies using immortalized rat proximal tubule cells revealed that 50 pmol/l TGF-beta1 disrupted albumin uptake (P < 0.001 vs. control), an inhibition significantly reversed by the use of the sTbetaRII.Fc (1,200 pmol/l). In vivo studies demonstrated that treatment with sTbetaRII.Fc reduced urinary albumin excretion by 36% at 4 weeks, 59% at 8 weeks (P < 0.001), and 45% at 12 weeks (P < 0.01 for diabetic vs. treated). This was correlated with an increase in megalin expression (P < 0.05 for diabetic vs. treated) and a reduction in collagen IV expression following sTbetaRII.Fc treatment (P < 0.001 for diabetic vs. treated). These changes occurred independently of changes in blood glucose levels. This study demonstrates that the sTbetaRII.Fc is a potential new agent for the treatment of fibrosis and albuminuria in diabetic nephropathy and may reduce albuminuria by reducing TGF-beta1-induced disruptions of renal proximal tubule cell uptake of albumin.     

Expression of interleukin 1-beta, transforming growth factor beta-1, and vascular endothelial growth factor in soft tissue over the implant before uncovering.

BACKGROUND: Overexpression of inflammatory cytokines interleukin 1-beta (IL-1beta), transforming growth factor beta-1 (TGF-beta1), and vascular endothelial growth factor (VEGF) may cause healing impairment following implant insertion, jeopardizing success especially in patients previously irradiated. Limited data is available regarding expression pattern of inflammatory cytokines in peri-implant soft tissue caused by the surgical intervention itself. STUDY DESIGN: This study examined 21 patients receiving dental implants. Biopsies of peri-implant tissue were harvested at re-entry 4 months after initial surgery. Eight patients underwent probing of untreated mucosa. Three groups were created (group 1: regular peri-implant mucosa; group 2: patients with irradiated peri-implant mucosa, radiation treatment due to oral squamous cell cancer; group 3: control). Immunohistochemical staining was performed for TGFss1, IL-1ss, and VEGF. RESULTS: Following the placement of dental implants (group 1 vs group 3) a significant increase (P > .05) in TGF-beta1, IL-1beta, and VEGF expression in the peri-implant mucosa was demonstrated. No alteration of this distinct pattern was found for previously irradiated tissue (group 1 vs. group 2). CONCLUSIONS: The study highlights the fundamental involvement of TGF-beta1, IL-1beta, and VEGF during the regeneration of peri-implant soft tissue structures. The use of extended interim solutions may be one clinical implication of these prolonged tissue remodeling processes.     

Interaction between active motion and exogenous transforming growth factor Beta during tibial fracture repair

OBJECTIVE: Evaluate the effects of axial motion and transforming growth factor beta (TGF-beta) on callus formation and fracture healing.DESIGN Prospective experimental design with a 39-day postfracture recovery. SETTING: Unrestricted cage activity with weight bearing as tolerated. ANIMALS: Twenty-two skeletally mature, female New Zealand White rabbits. INTERVENTIONS: Displaced, closed tibial fractures were reduced and stabilized in external fixators on the fourth day following fracture. Half of the fixators were locked for the duration of healing. The other fixators were locked for one week, then unlocked for the remaining four weeks. Half of the fractures in each fixator group received two injections of recombinant human TGF-beta1 (rhTGF-beta1). One injection was administered at the time of reduction, and the second was given 48 hours later. MAIN OUTCOME MEASUREMENTS: Interfragmentary axial motion was measured during floor activity. Biomechanical properties were measured during a torsion test to failure. Callus area and the distribution of tissues within the callus were determined by computer-aided histomorphometry. RESULTS: The administration of TGF-beta1 did not alter callus size, mechanical properties, or the distribution of tissues in the callus of fractures that were stabilized in locked external fixators. Recoverable axial motion fixation increased callus size, quantity of mineralized bone bridging the fracture, and maximum torque relative to locked fixation. The injection of TGF-beta1 negated the beneficial effects of axial motion by promoting the formation of a peripheral callus bridged by fibrous tissue rather than mineralized trabecular bone. CONCLUSIONS: Injection of rhTGF-beta1 during the first postfracture week does not provide a biologic boost that improves fracture healing. Injection of TGF-beta1 may be detrimental to healing under conditions when fracture motion is present. The results suggest that there is a tendency for exposure to TGF-beta1 to inhibit the normal development of peripheral callus in response to axial interfragmentary motion.     

Topical application of nerve growth factor improves fracture healing in rats

The aim of the present study was to examine the effects of nerve growth factor on the healing of unsplinted fractured ribs. After fracture of a rib in male rats, nerve growth factor was delivered by a miniosmotic pump to the fracture site for 7 days at the rate of 1.4 μg/day. Callus catecholamine concentrations, hone callus size, histomorphometry, and biomechanical properties of the repairing rib were measured at 7,21, and 42 days after fracture. After 21 days, concentrations of norepinephrine and epinephrine were significantly increased in the group treated with nerve growth factor compared with those in the control group (211% norepinephrine and 322% epinephrine). Also, the midline longitudinal area of non osseous (fibrous tissue and cartilage) callus of the fracture was significantly smaller (54%) and had a higher proportion of cartilage in the treated group than in the controls. By 42 days, there was only bony callus between the fracture ends in both the control group and the treated group. The treated group, however, again showed significantly elevated concentrations of norepinephrine and epinephrine (286 and 382%, respectively) and significantly elevated breaking stress (50%) and Young's modulus (51%), together wilh a reduction in the transverse cross-sectional area of the repair site (57%). The resultant increases in effectiveness and rate of repair of bone with administration of nerve growth factor suggest that it may play an important role in the healing processes of fractured bone.     

蛋白激酶C在高糖诱导近端小管上皮细胞细胞外基质及转化生长因子β1高表达中的作用

目的：探讨高糖作用下近端肾小管上皮细胞蛋白激酶C（PKC）活性的变化,以及PKC 激活对近端肾小管上皮细胞外细胞基质（ECM）及转化生长因子β1(TGF-β1）表达的调控作用。方法：采用LLC－PK1细胞株,将细胞分为正常对照组NG(5.5mmol/L D－葡萄糖）、高糖组HG（25mmol/L D-葡萄糖）、高渗组HM（25mmol/L甘露醇）、NG＋PKC抑制剂（PKCI）组（10μmol/L chelerythrine chloride)、HG＋PKCI组、HM＋PKCI组。分别检测各组细胞PKC活性,并运用原位杂交和免疫细胞化学法检测各组Ⅳ胶原（Co1Ⅳ）、纤连蛋白（FN）及TGF-β1mRNA和蛋白表达。结果：HG组细胞胞膜PKC活性较NG组升高3．3倍。HG组细胞Co1Ⅳ、FN及TGF－β1mRNA和蛋白水平均较NG组显著升高,HM组无此变化。高糖导致的ECM和TGF－β1高表达可被PKC抑制剂chelerythrine chloride所阻断。结论：由高糖诱导的近端小管上皮细胞ECM和TGF－β1高表达是通过激活PKC通路所介导。     

Local application of growth factors (insulin-like growth factor-1 and transforming growth factor-beta1) from a biodegradable poly(D,L-lactide) coating of osteosynthetic implants accelerates fracture healing in rats

In vitro and in vivo studies have demonstrated an osteoinductive effect of growth factors such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1). However, for therapeutic use in fracture treatment, questions remain with regard to the local application of these proteins. A controlled, local release of growth factors from a biodegradable polylactide coating of osteosynthetic implants may have a stimulating effect on fracture healing. Such implants could stabilize the fracture and their bioactive surface could function simultaneously as a local drug-delivery system. Previous studies have demonstrated the high mechanical stability of an approximately 10-14-microm-thick poly(D,L-lactide) (PDLLA) coating on metallic implants, which can even withstand the process of intramedullary insertion. Following an initial peak, 80% of incorporated growth factors IGF-1 and TGF-beta1 were continuously released within 42 days. The effect of locally applied IGF-1 and TGF-beta1 from a biodegradable PDLLA coating of intramedullary implants on fracture healing was investigated in a rat model. Midshaft fractures of the right tibia of 5-month-old female Sprague-Dawley rats (n = 127) were stabilized with coated vs. uncoated titanium Kirschner wires. X-ray examinations and blood analyses were performed, and body weight and body temperature measurements were taken throughout the experimental period. After 28 and 42 days, respectively, tibiae were dissected for mechanical torsional testing and histomorphometrical analyses. X-rays demonstrated an almost completely consolidated fracture, biomechanical testing showed a significantly higher maximum load and torsional stiffness, and histological and histomorphometric analyses demonstrated progressed remodeling after 28 and 42 days in the group treated with growth factors as compared with controls. Interestingly, the PDLLA coating itself revealed a positive effect on fracture healing even without incorporated growth factors. No systemic changes of serum parameters, including IGF-1 and IGF binding proteins, and no differences in body weight and body temperature were observed within and between groups. These findings suggest that the local application of growth factors from a biodegradable PDLLA coating of osteosynthetic implants accelerates fracture healing significantly without systemic side effects.     

Expression of transforming growth factor β isoforms and their roles in tendon healing

Transforming growth factor β (TGF‐β) plays active roles in tendon healing. However, the differential effects of TGF‐β isoforms on tendon healing have not been investigated. In cultured tendon fibroblasts, we tested the effects of TGF‐β1, β2, and β3 on the mRNA levels of COL1A1 and COL3A1 by quantitative real‐time polymerase chain reaction. We also investigated the expression of TGF‐β isoforms, TGF‐β receptors, procollagen Type I and Type III in a rat model of tendon healing. We found that TGF‐β3 exhibited the highest potency in stimulating COL1A1 and COL3A1. TGF‐β1 exerted antagonistic effects to TGF‐β2 and β3. All TGF‐β isoforms and procollagen Type I were confined to the edges of the healing tendon at day 28 postinjury. Our results indicated that interaction of TGF‐β isoforms exist in the regulation of collagen synthesis in tendon fibroblasts. Their effects may be further complicated by uneven spatial distribution of TGF‐β and TGF‐β receptors in healing tendons.     

Expression of the fibroblast growth factor receptor genes in fracture repair

The spatial and temporal expression domains of the fibroblast growth factor receptor genes were examined in the healing rat femur fracture by in situ hybridization. Fibroblast growth factor receptor gene expression was detected in diverse fracture tissues throughout healing. Fibroblast growth factor receptor 1 and 2 expression was present throughout fracture repair, in the early proliferating periosteal mesenchyme, in the osteoblasts during intramembranous bone formation, and in the chondrocytes and osteoblasts during endochondral bone formation. Fibroblast growth factor receptor 3 expression colocalized with fibroblast growth factor receptor 1 and 2 expression in the chondrocytes and osteoblasts beginning at 10 days of healing, and persisted throughout endochondral bone formation. Fibroblast growth factor receptor 3 recapitulated its expression in fetal skeletal development, suggesting that it has a similar function in the control of endochondral bone growth during fracture repair. Fibroblast growth factor receptor 4 expression was not observed at any time. The extensive colocalized expression of the fibroblast growth factor receptors in healing indicates that fibroblast growth factor regulation of fracture callus maturation is extensive, and accurate identification of the receptor isoforms is necessary to establish the functions of fibroblast growth factor family members in fracture repair.     

Interactions between extracellular matrix and growth factors in wound healing

Dynamic interactions between growth factors and extracellular matrix (ECM) are integral to wound healing. These interactions take several forms that may be categorized as direct or indirect. The ECM can directly bind to and release certain growth factors (e.g., heparan sulfate binding to fibroblast growth factor-2), which may serve to sequester and protect growth factors from degradation, and/or enhance their activity. Indirect interactions include binding of cells to ECM via integrins, which enables cells to respond to growth factors (e.g., integrin binding is necessary for vascular endothelial growth factor-induced angiogenesis) and can induce growth factor expression (adherence of monocytes to ECM stimulates synthesis of platelet-derived growth factor). Additionally, matrikines, or subcomponents of ECM molecules, can bind to cell surface receptors in the cytokine, chemokine, or growth factor families and stimulate cellular activities (e.g., tenascin-C and laminin bind to epidermal growth factor receptors, which enhances fibroblast migration). Growth factors such as transforming growth factor-β also regulate the ECM by increasing the production of ECM components or enhancing synthesis of matrix degrading enzymes. Thus, the interactions between growth factors and ECM are bidirectional. This review explores these interactions, discusses how they are altered in difficult to heal or chronic wounds, and briefly considers treatment implications.     

The experimental research of transforming growth factor-beta 1 expression during fracture healing]

OBJECTIVE: To observe the effect of TGF-beta 1 in the regulation of fracture healing. METHOD: The expression of transforming growth factor-beta 1 (TGF-beta 1) in different period of fracture healing was investigated by immunohistochemistry. RESULTS: It was found that the expression of TGF-beta 1 changed in different period. The cells in the cambial layer of the periostlum showed low or negative signal in the immediate injury response period. The osteoblasts differentiated from the periosteum cells stained strongly in the intramembranous ossification period, and the differentiated chondrocytes stained most strongly in the chondrogenesis period. The hypertrophic chondrocytes showed negative signal and the osteoblasts stained strongly in the endochondral ossification period. These results suggested that the expression of TGF-beta 1 was closely related to the proliferation and differentiation state of repair cells. CONCLUSION: TGF-beta 1 is intimately involved in the control of fracture healing.     

黄芩素对高糖诱导近端肾小管上皮细胞外基质及转化生长因子β1表达的影响

目的 探讨黄苓素对高糖诱导近端肾小管上皮细胞高度表达细胞外基质(ECM)及转化生长因子β1(TGF-β1)的干预作用。方法 LLC-PK1细胞分为(1)正常对照组(NG);(2)高糖组(HG);(3)HG+蛋白激酶抑制剂(PKCI)组(10μmol/L chelerythrine chloride);(4)HG+黄芩素组(50μmol/L);(5)HG+黄芩素组(100μmol/L);(6)HG+黄芩素组(200μmol/L)。检测各组PKC活性,并采用原位杂交法和细胞免疫化学技术(ABC法)分别检测细胞胶原(Col)Ⅳ、纤连蛋白(FN)及TGF-β1mRNA和蛋白的表达。结果 在高糖培养基中加入黄芩素100μmol/L及200μmol/L后可显著抑制细胞膜PKC活性(P<0.01),分别使细胞膜PKC活性下降42%、68%;200μmol/L黄芩素能使高糖组细胞ColⅣ、FN及TGF-β1 mRNA表达均显著下降,分别达55%、51%、46%,并显著抑制TGF-β1蛋白表达达51%(P<0.05)。结论 黄芩素可通过抑制细胞PKC活性而阻止高糖诱导近端小管细胞过度表达ECM及TGF-β1。