VEGF<Superscript>165</Superscript> and bFGF protein-based therapy in a slow release system to improve angiogenesis in a bioartificial dermal substitute in vitro and in vivo

BACKGROUND: Angiogenesis can be enhanced by several growth factors, like vascular endothelial growth factor-165 (VEGF(165)) and basic fibroblast growth factor (bFGF). Delayed release of such growth factors could be provided by incorporation of growth factors in fibrin matrices. In this study, we present a slow release system for VEGF(165) and bFGF in fibrin sealant. MATERIALS AND METHODS: In vitro: Pieces of Integratrade mark matrix of 15 mm in diameter were prepared. Integratrade mark matrices were divided into four groups (A=control; B=fibrin sealant; C=fibrin sealant+growth factors; D=growth factors). In vivo: The bioartificial dermal templates were transplanted into a full-skin defect of the back of nu-nu mice. Four different groups included each six matrices at 2 and 4 weeks. RESULTS: In vitro: In groups C and D, continuous release of VEGF(165) and bFGF was eminent. The incorporation of growth factors into fibrin sealant evoked a prolonged growth factor release (p < 0.05). In vivo: A significantly higher amount of vessels was quantified in groups C and D compared to groups A and B (p < 0.001). CONCLUSIONS: A model of slow protein release by combining VEGF(165) and bFGF with fibrin sealant was produced. This model resulted in a prolonged bioavailability of growth factors in vivo for functional purposes. Fibrin and collagen can release growth factors in vivo and induce significant and faster neovascularisation in bioartificial dermal templates.     

Expression of basic fibroblast growth factor mRNA in benign prostatic hyperplasia and prostatic carcinoma

In our previous work we demonstrated that prostate-derived growth factor (PrGF) is homologous to basic fibroblast growth factor (bFGF), not acidic fibroblast growth factor (aFGF). Using Northern blot analysis we now show that the messenger RNA for bFGF but not aFGF is expressed in benign prostatic hyperplastic (BPH) tissue as well as in carcinoma of the prostate (CAP). This not only corroborates our previous results, but suggests that PrGF is produced locally and not merely stored in the prostate. The demonstration of local production of bFGF by prostate tissue may indicate that this growth factor plays a role, either alone or in conjunction with other factors, in the etiology of benign hyperplasia or prostatic cancer.     

bFGF对关节软骨缺损的修复作用

目的观察碱性成纤维细胞生长因了对关节软骨缺损修复的影响。方法在兔双侧股骨髁间窝造成骨软骨缺损．一侧应用碱性成纤维细胞生长因子．另一侧作对照。术后3个月．利用组织切片及扫描电镜等方法，观察两侧骨软骨缺损修复情况。结果修复3个月后．对照侧软骨缺损难以完全修复．修复细胞形态多样，似成纤维细胞。蛋白多糖颗粒较少。应用碱性成纤维细胞生长因子删软骨缺损基本修复．修复细胞似软骨细胞．有较多的蛋白多糖颗粒与胶原纤维结合。缺损修复组织评分。碱性成纤维细胞生长因子组优于对照组。结论碱性成纤维细胞生长因子可促进骨软骨缺损的修复。     

Improved airway healing using basic fibroblast growth factor in a canine tracheal autotransplantation model.

OBJECTIVE: We studied 22 dogs to examine the effect of basic fibroblast growth factor (bFGF) alone, in comparison with omental or muscular wrapping on airway healing in a tracheal autotransplantation model. SUMMARY BACKGROUND DATA: Basic fibroblast growth factor is one of the most potent promoters of angiogenesis and has an ability to enhance blood supply to the ischemic airway. Topical administration of a fibrin glue enriched with 5 microg/cm2 bFGF, determined as a proportion of surface area of the tracheal grafts, improved revascularization of orthotopic canine tracheal autografts in a previous study. METHODS: All animals received orthotopic tracheal transplantation using 6-ring autografts that occupied a distal part of the thoracic trachea. Twenty-two animals were classified randomly into the following four groups: no treatment (Group G1, n = 4), muscular wrapping (Group G2, n = 4), omental wrapping (Group G3, n = 4), and topical administration of fibrin glue enriched with 5 microg/cm2 bFGF (Group G4, n = 10). Autografts were harvested 60 days after transplantation and assessed by the percent patency and histology. RESULTS: Devascularized tracheal autografts could not maintain their structural integrity without other treatments (Group G1). In contrast, more than half of all autografts receiving treatments remained viable, as demonstrated by gross and histologic findings (Groups G2, G3, and G4). Treatments with bFGF and omentum showed significantly better graft viability than no treatment. However, there was no statistical difference in the viability of tracheal autografts among the three treatment groups. In terms of the time performance ratio, bFGF was the best treatment for the devascularized autografts. CONCLUSIONS: Topical administration of bFGF was superior to the omental or muscular wrapping in terms of the time performance ratio. Clinical trials will be necessary to determine whether these findings are applicable to humans.     

Transforming growth factor-β2 (TGF-β2) reverses the inhibitory effects of fibrin sealant on cutaneous wound repair in the pig

Tensile strength of 2-cm, full-thickness, surgically incised porcine skin wounds sealed with fibrin sealant was enhanced compared to conventionally sutured wounds at 6 hours postwounding, but was significantly reduced after 3 days. Supplementation of fibrin sealant with transforming growth factor-beta2 (TGF-beta2) reversed the inhibitory effects of fibrin sealant on tensile strength at 3 days, and enhanced tensile strength at 7 days compared to suture or fibrin sealant alone. By 14 days, the tensile strengths of all wounds were similar, although wounds treated with fibrin sealant supplemented with TGF-beta2 showed a small, but statistically significant, improvement in wound strength compared to wounds treated with fibrin sealant alone. Histological assessment at day 7 revealed significant remnants of fibrin sealant at the wound site following fibrin sealant treatment alone, while wounds treated with fibrin sealant supplemented with TGF-beta2 or suture exhibited fibroblast infiltration and extracellular matrix deposition. At day 7, TGF-beta was immunolocalized in the base and margins of only wounds treated with fibrin sealant supplemented with TGF-beta2. A significant increase in matrix metalloproteinase-9 activity was found in fibrin sealant-treated wounds at day 7 as compared to sutured wounds. Addition of TGF-beta to the fibrin sealant suppressed the up-regulation of matrix metalloproteinase-9 in these wounds. These results suggest that fibrin sealant supplemented with TGF-beta may provide superior wound healing as compared to fibrin sealant alone.     

成纤维细胞生长因子和表皮生长因子及复合因子对兔ACL、MCL体外增殖作用

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor，aFGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)和表皮生长因子(epidermal growth factor，EGF)以及复合因子对兔前交叉韧带(anterior cruciate ligament，ACL)和内侧副韧带(medial collateral ligament，MCL)的促增殖作用。方法分离传代培养10周龄新西兰大白兔骨关节韧带的ACL和MCL的成纤维细胞，接种96孔板，并加入浓度为0(对照组)、1、5、10、50、100ng／ml的aFGF或bFGF，浓度为0(对照组)、1．56、3．13、6．25、12．50、25、50ng／ml的EGF，单独或aFGF EGF两种因子联用与细胞(n=4)共同培养48h，以XTT方法测定其促细胞增殖作用。结果3种生长因子单独应用对ACL和MCL都有促进作用，aFGF对两种细胞均存在量效关系；bFGF 1ng／ml，EGF 5ng／ml对ACL作用最好，而对MCL则是bFGF和EGF均存在量效关系。浓度为5ng／ml的aFGF与50ng／ml的EGF联合1ng／ml aFGF与3．13ng／mlEGF作用于ACL或MCL均有协同作用。结论3种生长因子对ACL和MCL均有促进作用，单独应用aFGF或联用EGF优于单一因子促进兔ACL和MCL细胞的增殖，并且提示低浓度的aFGF联用EGF优于单一生长因子。     

增生性瘢痕内3种成纤维细胞生长因子及其受体基因的表达

目的 探讨成纤维细胞生长因子(FGF)-7、酸性FGF(aFGF)、碱性FGF(bFGF)及其受体(FGFR1、FGFR2)在不同形成时期的增生性瘢痕中的基因表达。方法用病理学技术检测增生性瘢痕和正常皮肤的结构特征后,提取16例不同发生时期的增生性瘢痕和8例正常皮肤的总RNA后,分离mRNA,用逆转录-聚合酶链反应(RT-PCR)方法检测这5种基因在不同组织中的表达。结果在正常皮肤中,FGF-7,bFGF,FGFR1和FGFR2基因表达水平较低,而在增殖期的瘢痕中,这4种基因转录本的灰密度值分别为正常皮肤的2.1、2.1、3.6和2.8倍,基因表达显著增强(P<0.05);在成熟期的瘢痕中,FGF-7,FGFR1和FGFR2基因的表达量都低于增殖期的瘢痕,而bFGF仍保持高水平的基因表达。在正常皮肤和增殖期的瘢痕中,aFGF基因呈低水平表达,而在成熟期的瘢痕中aFGF基因表达明显增强(P<0.05)。结论FGF-7、bFGF及其受体基因表达升高可能是增生性瘢痕形成的机制之一,而FGF-7、FGFR1和FGFR2基因表达降低,aFGF表达增强可能与增生性瘢痕达到相对稳定的成熟期有关。     

烧伤创面应用生长因子后的DNA分析

将基因重组的人表皮细胞生长因子（ｒｈ－ＥＧＦ）、酸性成纤维细胞生长因子（ｒ－ａＦＧＦ）、碱性成纤维细胞生长因子（ｒ－ｂＦＧＦ）和牛脑提取液（ｂＢＥ）外用于烧伤创面，旨在探讨皮肤组织和细胞ＤＮＡ的含量及周期变化。实验结果显示：烧伤创面经应用ｒｈ－ＥＧＦ，ｒ－ａＦＧＦ，ｒ－ｂＦＧＦ和ｂＢＥ后第５天与生理盐水组（ＮＳ）相比，创面愈合率、表皮细胞和全厚层皮肤组织的ＤＮＡ含量均高于ＮＳ组（Ｐ＜０．０１）；全厚层皮肤ＤＮＡ的流式细胞仪分析表明，应用生长因子各组的Ｓ期细胞数百分比均高于ＮＳ组（Ｐ＜０．０１），细胞分裂增殖旺盛，Ｇ期细胞不断进入Ｓ期，使Ｓ期细胞数增加，ＤＮＡ的合成和含量增加。提示：烧伤创面外用细胞生长因子可促进表皮细胞的增殖，加速创面的愈合。     

烧伤创面应用生长因子后的DNA分析

将基因重组的人表皮细胞生长因子(rh-EGF)、酸性成纤维细胞生长因子(r-aFGF)、碱性成纤维细胞生长因子(r-bFGF)和牛脑提取液(bBE)外用于烧伤创面,旨在探讨皮肤组织和细胞 DNA 的含量及周期变化。实验结果显示:烧伤创面经应用 rh-EGF,r-aFGF,r-bFGF 和 bBE 后第5天与生理盐水组(NS)相比,创面愈合率、表皮细胞和全厚层皮肤组织的 DNA 含量均高于 NS 组(P<0.01);全厚层皮肤 DNA 的流式细胞仪分析表明,应用生长因子各组的 S 期细胞数百分比均高于 NS 组(P<0.01),细胞分裂增殖旺盛,G 期细胞不断进入 S 期,使 S 期细胞数增加,DNA 的合成和含量增加。提示:烧伤创面外用细胞生长因子可促进表皮细胞的增殖,加速创面的愈合。     

Basic fibroblast growth factor coating and endothelial cell seeding of a decellularized heparin-coated vascular graft

The objective of this study was to determine the effect of basic fibroblast growth factor (bFGF) coating on endothelial cell seeding and proliferation on a decellularized heparin coated vascular graft and to determine the retention of seeded cells on the graft under flow conditions. Disks of heparin coated decellularized grafts were incubated for 24 h as controls or with bFGF. Human microvascular endothelial cells (HMECs) or canine peripheral blood endothelial progenitor cells (CEPC) were seeded onto the disks and incubated for 96 h or 48 h, respectively. HMECs were also seeded onto the luminal surfaces of two heparin-coated decellularized grafts for 3 h. One graft was placed in a perfusion culture system and cultured for an additional 6 h with flow and pressure. After culturing, there were 4.7 +/- 1.4 cells/mm(2) HMECs on control grafts and 11.4 +/- 1.4 cells/mm(2) in bFGF treated grafts (P < 0.05). Likewise, with CEPCs, there were 14.8 +/- 4.8 cells/mm(2) in control grafts and 33.3 +/- 7.3 cells/mm(2) in bFGF treated grafts. After only 3 h of cell attachment, 60% of HMECs were retained in the intact graft exposed flow relative to the static control graft, which is an acceptable level. These data demonstrate that bFGF coating on the heparin bound decellularized grafts significantly increases both HMEC and dog EPC proliferation and that seeded cells are stable under perfusion conditions.     

Failure of airway healing in an ovine autotransplantation model that includes basic fibroblast growth factor

OBJECTIVE: Basic fibroblast growth factor is among the most potent promoters of angiogenesis. Its ability to enhance the blood supply to ischemic airways or nonvascularized tracheal autograft has been demonstrated. Its cumulative effect with muscular wrapping and its efficacy in a noncanine large animal model remain unknown. Treatment with basic fibroblast growth factor and muscular wrapping were compared with no special treatment and with muscular wrapping alone in an ovine tracheal autotransplantation model. METHODS: All sheep underwent orthotopic tracheal transplantation with 5 to 8 ring autografts in the cervical trachea. Fifteen sheep were classified randomly into the following three groups: no treatment (group A, n = 5), muscular wrapping with the right sternomastoid muscle (group B, n = 5), and topical administration of fibrin glue enriched with 2 microg/cm(2) basic fibroblast growth factor (group C, n = 5). RESULTS: Devascularized tracheal autografts were unable to maintain their structural integrity without other treatment (group A). However, the grafts were surrounded by well-vascularized connective tissue. In the muscular wrapping group (group B), infections occurred around the grafts, and the muscular wrapping was subject to necrosis. No neovascularization of the grafts occurred. Therapy with basic fibroblast growth factor (group C) led to improved muscular wrapping circulation and to adherence to the tracheal stumps. However, no success was achieved in validating the circulation in the grafts. CONCLUSIONS: In contrast to the results achieved by other authors with canine models, the neovascularization of tracheal autografts was not achieved in sheep with the topical administration of basic fibroblast growth factor. Cranially pediculated muscular wrapping led to poorer circulation in the tissue around the graft than did no therapy at all.     

Repair of cartilage defects with periosteal grafts.

Alternative sources for repair of cartilage defects are limited and donor sites are associated with morbidity. It is known that cartilage development from periosteal grafts is possible. Various factors have been found positively to affect this process in experimental settings. However, all of these studies were limited to joint cartilage. We conducted an experimental study in rabbits for the investigation of the elastic cartilage regeneration from perichondrial and periosteal grafts together with the effects of hyaluronan on this process. 1 x 1 cm(2) cartilage defects were created on the elastic ear cartilage of rabbits. Four experimental groups with 18 ears in each group were created: Group 1 (repair with perichondrium graft), group 2 (repair with periosteum graft), group 3 (repair with periosteum graft+hyaluronan), group 4 (defect-only control group). Macroscopic and microscopic evaluations were done on the 4th, 8th and 12th weeks. Cellular morphology of the regenerated cartilage and its integration and similarity with adjacent cartilage were evaluated. Cartilage regeneration groups 1, 2 and 3 were found to be statistically different from the control group. There was not a significant difference between groups 1 and 2 or 2 and 3. There is no significant difference between perichondrial and periosteal grafts in cartilage regeneration, and hyaluronan has no beneficial effect on this process.     

Effects of acid and basic fibroblast growth factor and heparin on resorption of cultured fetal rat long bones.

We tested acid and basic fibroblast growth factor (aFGF and bFGF), members of the heparin binding FGF family, for their ability to stimulate bone resorption as measured by the release of previously incorporated 45Ca from cultured fetal rat long bones in the presence and absence of heparin. Purified low-molecular-weight heparin (LMW heparin) at 5-125 micrograms/ml had no direct stimulatory effect. There was little effect from aFGF (10(-11)-10(-8) M) alone, but increased resorption was observed in the presence of LMW heparin. With bFGF, increased bone resorption was observed at 10(-9) M but not at 10(-8) M. The stimulatory effects of aFGF and bFGF in the presence of LMW heparin were not blocked by the addition of indomethacin (10(-6) M), which blocks prostaglandin production, or hydroxyurea (10(-3) M), which blocks DNA synthesis. However, pretreatment with aphidicolin (3 x 10(-5) M), a potent inhibitor of DNA synthesis, blocked the effect of acid FGF and diminished the effect of bFGF. These results indicate that both aFGF and bFGF can stimulate bone resorption by a prostaglandin-independent mechanism, particularly in the presence of heparin. The activation of FGF-mediated bone resorption by heparin could play a role in producing the osteoporosis that has been described with heparin therapy and mastocytosis.     

The iliac crest cartilaginous cap

Bone and cartilage grafts can be procured from the ilium either separately or as composite chondroosseous grafts when sufficient cartilage is present. The thickness and anatomy of this iliac cartilaginous cap was analyzed in relationship to age in 50 individuals. Histology was that of normal hyaline cartilage. The cartilage alone was more pliable with little memory when compared with auricular or septal cartilage. The cartilage/bone junction was very strong. Cartilage thickness ran from close to 1 cm at age 5 to a diminished zero at age 25.     

The use of photooxidized, mushroom-structured osteochondral grafts for cartilage resurfacing--a comparison to photooxidized cylindrical grafts in an experimental study in sheep

OBJECTIVE: This article addresses the problem of structural design with osteochondral grafts used for cartilage resurfacing. METHODS: Photooxidized cylindrical or mushroom-shaped grafts were surgically implanted in the weight bearing area of the medial and lateral femoral condyles of eight sheep (condyles: N=8/group). Both types of photooxidized grafts contained no viable chondrocytes at the time of implantation. Results were evaluated at 2 and 6 months after surgical implantation of the grafts. Qualitative and quantitative evaluation of the subchondral bone area was performed using plastic embedded sections of non-decalcified bone and cartilage specimens and placing emphasis on graft anchorage, cyst-like lesions at the base of the cartilage junction and at the base of the graft in the subchondral bone region. Cartilage morphology was studied qualitatively focusing on viability of the graft and adjacent host cartilage, while a score system was developed for semi-quantitative evaluation of the overall articular cartilage performance. The semiquantitative scores and histomorphometrical measurements were subjected to statistical analysis using a factorial analysis of variance (ANOVA-test). RESULTS: The photooxidized mushroom-shaped grafts developed less fibrous tissue and cyst-like lesions in the subchondral bone area at 2 and 6 months compared to the cylindrical grafts. Areas of endochondral ossification and bone remodeling were noticeable in the mushroom structured grafts at 2 months, and also bone remodeling was more complete at 6 months than with the cylindrical grafts. Increased numbers of cells were seen in the basal remodeling zones of both graft types increased from the 2 months to the 6 months specimens, but mushroom structured grafts showed better results. In both graft types, however, the midzone of the cartilage matrix was still acellular at 6 months. Cells from the subchondral bone area started to penetrate the calcified cartilage zone and tide mark at 2 months and repopulated the old photooxidized cartilage matrix already at 6 months after implantation. Cartilage repopulation was dependent on a stable subchondral bone area in both types of grafts. Matrix degradation of the adjacent host cartilage was minimal at 2 and 6 months. At 6 months a junction between host and graft cartilage was already noticed in some of the mushroom-shaped grafts. CONCLUSION: This study confirmed the importance of the subchondral bone area for osteochondral graft survival. In addition it demonstrated that the structure of the graft influences considerably the architecture of the subchondral bone, and with this the possibility for the repopulation of the old cartilage matrix including the junction between the host and graft cartilage matrix.     

Skin grafting with fibrin glue in burns

Summary In an attempt to obtain better function and appearance minimize blood loss and increase graft survival with minimal postoperative care, deep burns are treated by applying the fibrin glue to difficult recipient sites. A two-component fibrin sealant is used, fibrinogen made from pooled human plasma and bovine thrombin (Tissucol, Immuno AG, Vienna, Austria). The fibrin sealant is described as well as the technique used to seal the skin grafts in 15 patients (18 procedures). Indications for graft sealing are limited to grafts over areas subject to movement (joints, face, neck, hands) and to non-meshed grafts for cosmetic reasons. This clinical experience demonstrates several advantages of sealing skin grafts on burn wounds: hemostasis, early adherence and wound healing are improved; better cosmetic results are obtained with sheet grafts on the face and neck; operating time is reduced; no special dressings are required; physiotherapy starts after 24 h; the period of rehabilitation is reduced as well as the incidence of secondary procedures.     

Healing of full-thickness defects of the articular cartilage in rabbits using fibroblast growth factor-2 and a fibrin sealant

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant. Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect. Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.     

Effects of combination therapy using basic fibroblast growth factor and mature adipocyte-derived dedifferentiated fat (DFAT) cells on skin graft revascularisation

Abstract

Background. Although the benefits of basic fibroblast growth factor (bFGF) for wound healing and angiogenesis are well known, its effects on the process of skin graft revascularisation have not been clarified. It was hypothesised that bFGF would be beneficial to promote taking of skin grafts, but that the effect might be limited in the case of bFGF monotherapy. Therefore, this study investigated the efficacy of combination therapy using bFGF and dedifferentiated fat (DFAT) cells. DFAT cells have multilineage differentiation potential, including into endothelial cells, similar to the case of mesenchymal stem cells (MSC). Methods. Commercially available human recombinant bFGF was used. DFAT cells were prepared from SD strain rats as an adipocyte progenitor cell line from mature adipocytes. Full-thickness skin was lifted from the back of SD strain rats and then grafted back to the original wound site. Four groups were established prior to skin grafting: control group (skin graft alone), bFGF group (treated with bFGF), DFAT group (treated with DFAT cells), and combination group (treated with both bFGF and DFAT cells). Tissue specimens for histological examination were harvested 48 hours after grafting. Results. The histological findings for the bFGF group showed vascular augmentation in the grafted dermis compared with the control group. However, the difference in the number of revascularised vessels per unit area did not reach statistical significance against the control group. In contrast, in the combination group, skin graft revascularisation was significantly promoted, especially in the upper dermis. Conclusion. The results suggest that replacement of the existing graft vessels was markedly promoted by the combination therapy using bFGF and DFAT cells, which may facilitate skin graft taking.