Growth Factor Delivery for Tissue Engineering

A tissue-engineered implant is a biologic-biomaterial combination in which some component of tissuehas been combined with a biomaterial to create a device for the restoration or modification of tissue ororgan function. Specific growth factors, released from a delivery device or from co-transplanted cells,would aid in the induction of host paraenchymal cell infiltration and improve engraftment of co-deliveredcells for more efficient tissue regeneration or ameliorate disease states. The characteristic properties ofgrowth factors are described to provide a biological basis for their use in tissue engineered devices. Theprinciples of polymeric device development for therapeutic growth factor delivery in the context of tissueengineering are outlined. A review of experimental evidence illustrates examples of growth factor deliveryfrom devices such as micropaticles, scaffolds, and encapsulated cells, for their use in the applicationareas of musculoskeletal tissue, neural tissue, and hepatic tissue.     

Polymeric Growth Factor Delivery Strategies for Tissue Engineering

Purpose. Tissue engineering seeks to replace and regrow damaged or diseased tissues and organs from either cells resident in the surrounding tissue or cells transplanted to the tissue site. The purpose of this review is to present the application of polymeric delivery systems for growth factor delivery in tissue engineering. Methods. Growth factors direct the phenotype of both differentiated and stem cells, and methods used to deliver these molecules include the development of systems to deliver the protein itself, genes encoding the factor, or cells secreting the factor. Results. Results in animal models and clinical trials indicate that these approaches may be successfully used to promote the regeneration of numerous tissue types. Conclusions. Controlling the dose, location, and duration of these factors through polymeric delivery strategies will dictate their utility in tissue regeneration.     

Bioactive Electrospun Scaffolds Delivering Growth Factors and Genes for Tissue Engineering Applications

A biomaterial scaffold is one of the key factors for successful tissue engineering. In recent years, an increasing tendency has been observed toward the combination of scaffolds and biomolecules, e.g. growth factors and therapeutic genes, to achieve bioactive scaffolds, which not only provide physical support but also express biological signals to modulate tissue regeneration. Huge efforts have been made on the exploration of strategies to prepare bioactive scaffolds. Within the past five years, electrospun scaffolds have gained an exponentially increasing popularity in this area because of their ultrathin fiber diameter and large surface-volume ratio, which is favored for biomolecule delivery. This paper reviews current techniques that can be used to prepare bioactive electrospun scaffolds, including physical adsorption, blend electrospinning, coaxial electrospinning, and covalent immobilization. In addition, this paper also analyzes the existing challenges (i.e., protein instability, low gene transfection efficiency, and difficulties in accurate kinetics prediction) to achieve biomolecule release from electrospun scaffolds, which necessitate further research to fully exploit the biomedical applications of these bioactive scaffolds.     

Injectable Biodegradable Polyurethane Scaffolds with Release of Platelet-derived Growth Factor for Tissue Repair and Regeneration

Purpose  The purpose of this work was to investigate the effects of triisocyanate composition on the biological and mechanical properties of biodegradable, injectable polyurethane scaffolds for bone and soft tissue engineering. Methods  Scaffolds were synthesized using reactive liquid molding techniques, and were characterized in vivo in a rat subcutaneous model. Porosity, dynamic mechanical properties, degradation rate, and release of growth factors were also measured. Results  Polyurethane scaffolds were elastomers with tunable damping properties and degradation rates, and they supported cellular infiltration and generation of new tissue. The scaffolds showed a two-stage release profile of platelet-derived growth factor, characterized by a 75% burst release within the first 24 h and slower release thereafter. Conclusions  Biodegradable polyurethanes synthesized from triisocyanates exhibited tunable and superior mechanical properties compared to materials synthesized from lysine diisocyanates. Due to their injectability, biocompatibility, tunable degradation, and potential for release of growth factors, these materials are potentially promising therapies for tissue engineering.     

Nanoparticulate Systems for Growth Factor Delivery

The field of nanotechnology, which aims to control and utilize matter generally in 1–100 nm range, has been at the forefront of pharmaceutical development. Nanoparticulate delivery systems, with their potential to control drug release profiles, prolonging the presence of drugs in circulation, and to target drugs to a specific site, hold tremendous promise as delivery strategies for therapeutics. Growth factors are endogenous polypeptides that initiate intracellular signals to regulate cellular activities, such as proliferation, migration and differentiation. With improved understanding of their roles in physiopathology and expansion of their availability through recombinant technologies, growth factors are becoming leading therapeutic candidates for tissue engineering approaches. However, the outcome of growth factor therapeutics largely depends on the mode of their delivery due to their rapid degradation in vivo, and non-specific distribution after systemic administration. In order to overcome these impediments, nanoparticulate delivery systems are being harnessed for spatiotemporal controlled delivery of growth factors. This review presents recent advances and some disadvantages of various nanoparticulate systems designed for effective intact growth factor delivery. The therapeutic applications of growth factors delivered by such systems are reviewed, especially for bone, skin and nerve regeneration as well as angiogenesis. Finally, future challenges and directions in the field are presented in addition to the current limitations.     

Elastase-Sensitive Elastomeric Scaffolds with Variable Anisotropy for Soft Tissue Engineering

Purpose  To develop elastase-sensitive polyurethane scaffolds that would be applicable to the engineering of mechanically active soft tissues. Methods  A polyurethane containing an elastase-sensitive peptide sequence was processed into scaffolds by thermally induced phase separation. Processing conditions were manipulated to alter scaffold properties and anisotropy. The scaffold’s mechanical properties, degradation, and cytocompatibility using muscle-derived stem cells were characterized. Scaffold in vivo degradation was evaluated by subcutaneous implantation. Results  When heat transfer was multidirectional, scaffolds had randomly oriented pores. Imposition of a heat transfer gradient resulted in oriented pores. Both scaffolds were flexible and relatively strong with mechanical properties dependent upon fabrication conditions such as solvent type, polymer concentration and quenching temperature. Oriented scaffolds exhibited anisotropic mechanical properties with greater tensile strength in the orientation direction. These scaffolds also supported muscle-derived stem cell growth more effectively than random scaffolds. The scaffolds expressed over 40% weight loss after 56 days in elastase containing buffer. Elastase-sensitive scaffolds were complete degraded after 8 weeks subcutaneous implantation in rats, markedly faster than similar polyurethanes that did not contain the peptide sequence. Conclusion  The elastase-sensitive polyurethane scaffolds showed promise for application in soft tissue engineering where controlling scaffold mechanical properties and pore architecture are desirable.     

Local Extravascular Growth Factor Delivery in Myocardial Ischemia

A number of angiogenic growth factors have been demonstrated in vivo to promote angiogenesis in ischemic myocardium, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). We used the porcine ameroid constrictor model to simulate chronic ischemia in an effort to study the role of exogenous growth factor delivery in the development of coronary collateral circulation. Heparin alginate microspheres were used to deliver bFGF, while an implantable osmotic pump was used for VEGF delivery.

Anti-von Willebrand antibody staining was used to visualize microvessels in the porcine heart sections obtained from both ischemic and non-ischemic myocardium. A significant increase in the number of microvessels in growth factor-treated pigs compared to control animals was noted. Myocardial blood flow was used to determine the physiologic impact of these findings. In bFGF and VEGF treated animals resting collateral flow values significantly exceeded those of the control group. In addition, both bFGF and VEGF administration resulted in improvements in myocardial perfusion sufficient to prevent stress-induced deterioration of myocardial performance. Results of the studies demonstrate that local perivascular drug delivery can be effectively used to induce angiogenesis in chronically ischemic myocardium.     

Heparin Immobilized Porous PLGA Microspheres for Angiogenic Growth Factor Delivery

Purpose Heparin immobilized porous poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres were prepared for sustained release of basic fibroblast growth factor (bFGF) to induce angiogenesis.Materials and Methods Porous PLGA microspheres having primary amine groups on the surface were prepared using an oil-in-water (O/W) single emulsion method using Pluronic F-127 as an extractable porogen. Heparin was surface immobilized via covalent conjugation. bFGF was loaded into the heparin functionalized (PLGA-heparin) microspheres by a simple dipping method. The bFGF loaded PLGA-heparin microspheres were tested for in vitro release and in vivo angiogenic activity.Results PLGA microspheres with an open-porous structure were formed. The amount of conjugated amine group onto the microspheres was 1.93 ± 0.01 nmol/mg-microspheres, while the amount of heparin was 95.8 pmol/mg-microspheres. PLGA-heparin microspheres released out bFGF in a more sustained manner with a smaller extent of initial burst than PLGA microspheres, indicating that surface immobilized heparin controlled the release rate of bFGF. Subcutaneous implantation of bFGF loaded PLGA-heparin microspheres in mice significantly induced the formation of new vascular microvessels.Conclusions PLGA microspheres with an open porous structure allowed significant amount of heparin immobilization and bFGF loading. bFGF loaded PLGA-HP microspheres showed sustained release profiles of bFGF in vitro, demonstrating reversible and specific binding of bFGF to immobilized heparin. They also induced local angiogenesis in vivo in an animal model.     

基于海洋多糖水凝胶的组织工程材料研究应用进展

海洋天然多糖以其优异的生物相容性、生物可降解性、安全性以及特定的生物活性,成为生物医用材料研究的热点领域之一.近年来,基于海洋来源的糖类生物大分子开发的新型水凝胶在组织工程领域得到了广泛应用.本文综述了基于海藻酸盐、壳聚糖、透明质酸、卡拉胶和岩藻聚糖硫酸酯研发的功能性水凝胶,评述了这些水凝胶的设计思路、制备方法、理化性...     

Intracranial Delivery of Recombinant Nerve Growth Factor: Release Kinetics and Protein Distribution for Three Delivery Systems

Purpose. Three different polymeric delivery systems, composed of either poly(ethylene-co-vinyl acetate) (EVAc) or poly(lactide-co-gly-colide) (PLGA), were used to administer recombinant human nerve growth factor (rhNGF) intracranially in rats. Methods. The delivery systems were characterized with respect to release kinetics, both in the brain and in well-stirred buffer solutions. Results. During incubation in buffered saline, the delivery systems released rhNGF in distinct patterns: sustained (EVAc), immediate (PLGA₁), and delayed (PLGA₂). One 10-mg delivery system was implanted in each rat and an ELISA technique was used to determine the amount of rhNGF in 1-mm coronal brain slices produced immediately after removal of the delivery system. High levels of rhNGF (as high as 60,000 ng in a brain slice of 50 L) were recovered from the brain tissue at 1,2, and 4 weeks after implantation. With all three delivery systems, the amount of rhNGF in each brain slice decreased exponentially with distance from the implant site; the distance over which concentration decreased by 10-fold was 2–3 mm for all delivery systems. When rhNGF release was moderate (10 to 200 ng rhNGF/ day), the total amount of rhNGF in the brain increased linearly with release rate, suggesting an overall rate of rhNGF elimination of 0.4 hr^–1 or a half-life of 1.7 hr. With higher release rates (500 to 50,000 ng rhNGF/day), total amounts of rhNGF in the brain were considerably higher than anticipated based on this rate of elimination. Conclusions. Polymeric controlled release can provide high, localized doses of rhNGF in the brain. All of the experimental data were consistent with penetration of rhNGF through the brain tissue with a diffusion coefficient 8 X 10^–7 cm²/s, which is 50% of the diffusion coefficient in water.     

Vascular Endothelial Growth Factor Gene Delivery for Revascularization in Transplanted Human Islets

PURPOSE: Islet transplantation is limited by islet graft failure because of poor revascularization, host immune rejection, and nonspecific inflammatory response. Human vascular endothelial growth factor (hVEGF) gene delivery is likely to promote islet revascularization and survival. METHODS: We evaluated gene expression from a bicistronic plasmid encoding hVEGF and enhanced green fluorescent protein (EGFP) (pCMS-EGFP-hVEGF). Glucose responsiveness of islets was evaluated both in vitro and in vivo, and revascularization in islet graft was evaluated by immunohistochemistry. RESULTS: After transfection, hVEGF and EGFP expression levels were comparable with original monocistronic plasmids in Jurkat cells but higher and prolonged hVEGF expression in islets transfected with the bicistronic plasmid was observed, possibly as the result of differences in promoter strength and hypoxia response. The 3:1 w/w complexes showed little toxicity to islets at a dose of 5 microg DNA per 2000 islets. On glucose challenge, insulin release from transfected islets as well as secretion from islets after transplantation under the mouse kidney capsules in response to glucose stimulation, increased with time. Immunohistochemical staining of transplanted islets using mouse anti-human insulin, mouse anti-human von Willebrand factor, and rat anti-mouse CD31 antibodies suggests that islets are functional and there is new blood vessel formation. CONCLUSIONS: These findings suggest that transient hVEGF gene expression by the islets may promote islet revascularization and prolong islet survival after transplantation.     

结缔组织生长因子在单侧肾静脉结扎大鼠肾组织中的表达

目的:探讨结缔组织生长因子(CTGF)在单侧肾静脉结扎大鼠肾组织中的表达及与肾小管上皮细胞表型转化的关系.方法:雄性Wistar大鼠50只,随机均分为模型组和对照组,模型组25只行左侧肾静脉结扎术,分别于术后5、10、15、20、25d处死大鼠(每组5只).对照组只分离但不结扎.模型组大鼠取双侧肾脏行HE和Masson染色,观察各时间点肾小管间质的改变;采用免疫组化SABC法检测角蛋白(CK)、CTGF和α-平滑肌肌动蛋白(α-SMA)在肾组织内的表达和分布;应用计算机图像分析系统对各项结果进行半定量分析.结果:随结扎时间的延长,模型组大鼠小管间质纤维化逐渐加重,且CTGF、α-SMA的表达逐渐增加;CTGF与α-SMA的表达呈正相关;二者与胶原蛋白的表达均呈正相关.结论:肾静脉结扎导致CTGF的表达增加,CTGF可能通过促进间质中肌成纤维细胞的形成而参与肾间质纤维化.     

结缔组织生长因子与恶性肿瘤的研究进展

临床上,80%以上的恶性肿瘤病人死于肿瘤的侵袭和转移,可见恶性肿瘤的侵袭和转移是导致治疗失败和死亡的主要原因。随着分子生物学的进展和对肿瘤发生遗传机制的进一步了解,以及对恶性肿瘤发生、发展过程和预后的细致深入研究,人们发现恶性肿瘤的发生、发展是一个多阶段多因素共同参与的生物学过程。     

来氟米特对糖尿病模型大鼠肾组织中血小板衍化生长因子的影响研究

目的:探讨来氟米特对糖尿病模型大鼠肾组织中血小板衍化生长因子(PDGF)的影响。方法:取大鼠单剂量注射链脲佐菌素60mg·kg-1建立糖尿病模型,建模成功后分为模型组(自来水)和治疗组(来氟米特,5mg·kg-1·d-1),每组16只,灌胃给予相应药物,于第4、8周末各组随机取样8只,检测血糖、胆固醇、甘油三酯、血肌酐、尿白蛋白、尿β2-微球蛋白(β2-MG)等生化指标及肾脏肥大指标的变化,并以免疫组化法检测肾组织中PDGF表达。另设正常对照组进行比较。结果:与模型组比较,治疗组大鼠第4周除血糖外、第8周除甘油三酯外,其他生化指标、肾脏肥大指标以及肾组织中PDGF表达均降低。结论:来氟米特可能通过下调糖尿病模型大鼠肾组织中PDGF表达来实现肾脏保护作用。     

结缔组织生长因子在肺间质纤维化发病机制中的地位和作用

为了解结缔组织生长因子 (connective tissue growth factor,CTGF)在大鼠肺纤维化模型中的表达状况及其在肺纤维化发病机制中的作用 ,将 42只 Wistar大鼠随机分为模型组 (M组 )和对照组 (C组 ) ,M组气管内灌注博莱霉素 ,C组气管内灌注生理盐水。于灌注后第 7、14、2 8天每组分别处死大鼠 7只 ,制备大鼠肺组织切片。用免疫组化 SABC法分别检测两组大鼠肺组织中 CTGF及转化生长因子β1 (TGF-β1 )、 型胶原纤维 (Collagen )的蛋白在肺内的表达水平。结果显示 ,CTGF作为 TGF -β1 的下游因子 ,可能通过促进细胞外基质 (ECM)如胶原蛋白等合成而在博莱霉素诱导的肺纤维化形成过程中起重要作用     

Designing 3D Photopolymer Hydrogels to Regulate Biomechanical Cues and Tissue Growth for Cartilage Tissue Engineering

PURPOSE: Synthetic hydrogels fabricated from photopolymerization are attractive for tissue engineering for their controlled macroscopic properties, the ability to incorporate biological functionalities, and cell encapsulation. The goal of the present study was to exploit the attractive features of synthetic hydrogels to elucidate the role of gel structure and chemistry in regulating biomechanical cues. METHODS: Cartilage cells were encapsulated in poly(ethylene glycol) (PEG) hydrogels with different crosslinking densities. Cellular deformation was examined as a function of gel crosslinking. The effects of continuous versus intermittent dynamic loading regimens were examined. RGD, a cell adhesion peptide, was incorporated into PEG gels and subjected to mechanical loading. Chondrocyte morphology and activity was assessed by anabolic and catabolic ECM gene expression and matrix production by collagen and glycosaminoglycan production. RESULTS: Cell deformation was mediated by gel crosslinking. In the absence of loading, anabolic activity was moderately upregulated while catabolic activity was significantly inhibited regardless of gel crosslinking. Dynamic loading enhanced anabolic activities, but continuous loading inhibited catabolic activity, while intermittent loading stimulated catabolic activity. RGD acted as a mechanoreceptor to influence tissue deposition. CONCLUSIONS: We demonstrate the ability to regulate biomechanical cues through manipulations in the gel structure and chemistry and cartilage tissue engineering.     

Delivery of Nerve Growth Factor to Brain Via Intranasal Administration and Enhancement of Brain Uptake

The main objective of the study was to investigate the efficacy of chitosan to facilitate brain bioavailability of intranasally administered nerve growth factor (NGF). In vitro permeability studies and electrical resistance studies were carried out across the bovine olfactory epithelium using Franz diffusion cells. The bioavailability of intranasally administered NGF in rat hippocampus was determined by carrying out brain microdialysis in Sprague–Dawley rats. The in vitro permeation flux across the olfactory epithelium of NGF solution without chitosan (control) was found to be 0.37 ± 0.06 ng/cm²/h. In presence of increasing concentration of chitosan (0.1%, 0.25%, and 0.5%, w/v) the permeation flux of NGF was found to be 2.01 ± 0.12, 3.88 ± 0.19, and 4.12 ± 0.21 ng/cm²/h respectively. Trans-olfactory epithelial electrical resistance decreased ～34.50 ± 4.06% in presence of 0.25% (w/v) chitosan. The C_max in rats administered with 0.25% (w/v) chitosan and NGF was 1008.62 ± 130.02 pg/mL, which was significantly higher than that for rats administered with NGF only 97.38 ± 10.66 pg/mL. There was ～14-fold increase in the bioavailability of intranasally administered NGF with chitosan than without chitosan. Chitosan can enhance the brain bioavailability of intranasally administered NGF. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3640–3646, 2009     

Local and Sustained Vascular Endothelial Growth Factor Delivery for Angiogenesis Using an Injectable System

Purpose  We hypothesize that a microsphere/hydrogel combination system could be useful for the local and sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) to enhance angiogenesis in vivo. Methods  Poly(d,l-lactide-co-glycolide) (PLGA) microspheres containing rhVEGF were loaded into alginate gels by ionic cross-linking. The rhVEGF release from the system was monitored and bioactivity was tested in vitro. The combination system was subcutaneously injected into mice using a syringe, and new blood vessel formation was evaluated. Results  Sustained rhVEGF release from the combination system was observed for 3 weeks, and the released rhVEGF remained bioactive. Endothelial cell proliferation was significantly enhanced when cells were cultured with the rhVEGF-releasing combination system in vitro. When the combination system was implanted, the granulation tissue layer was thicker with more newly formed blood vessels than that with a single dose VEGF injection. Conclusion  The rhVEGF release was controlled by varying relative portions of microspheres and hydrogels in combination delivery systems, which efficiently promoted new blood vessel formation in vivo. This combination system could be a promising delivery vehicle for therapeutic angiogenesis.