Preparation,in vitro release,in vivo absorption and biocompatibility studies of insulin-loaded microspheres in rabbits

The purpose of this study was to develop a single-dose insulin delivery system based on poly (lactide-co-glycolide) (PLGA) microspheres to provide basal insulin level for a prolonged period. Insulin-loaded PLGA microspheres were prepared by water-in-oil-in-water double emulsion (batch A) and solid-in-oil-in-water emulsion (batch B) methods. Microspheres were characterized for physical characteristics and in vitro release. In vivo absorption of insulin and biocompatibility of insulin-loaded PLGA microspheres were performed in diabetic New Zealand white rabbits. Light and transmission electron microscopy were performed on the skin tissues excised from microspheres injected sites in order to study the biocompatibility. The burst release of insulin was high (47%) from batch B and low (5%) from batch A. Therefore, we mixed microspheres of batch A and B in ratio of 3:1 w/w, which produced desirable in vitro release profile. In vivo absorption study showed that insulin-loaded microspheres provided a serum insulin level of 20-40 microU/ml up to 40 days. Biocompatibility study provided evidence of normal inflammatory and foreign body reactions, which were characterized by the presence of macrophages, fibroblasts and foreign body giant cells. Neither necrosis nor tissue damage was identified. At the end of 12 weeks, no distinct histological differences were observed in comparison to the control tissue samples. In conclusion, insulin-loaded PLGA microspheres controlled the in vivo absorption of insulin to maintain the basal insulin level for longer period and the delivery system was biocompatible.     

聚丙交酯- 乙交酯降解研究进展

本文从分子量、孔径大小和孔径率、力学性能等方面介绍了研究聚丙交酯-乙交酯降解行为的方法,综述了聚丙交酯-乙交酯的化学水解机理和酶催化水解机理,影响聚丙交酯-乙交酯降解速率的内外因素,并比较了聚丙交酯-乙交酯体内外降解的异同,对未来聚丙交酯-乙交酯降解研究的方向提出了展望。     

聚丙交酯-乙交酯降解研究进展

本文从分子量、孔径大小和孔径率、力学性能等方面介绍了研究聚丙交酯-乙交酯降解行为的方法,综述了聚丙交酯-乙交酯的化学水解机理和酶催化水解机理,影响聚丙交酯-乙交酯降解速率的内外因素,并比较了聚丙交酯-乙交酯体内外降解的异同,对未来聚丙交酯-乙交酯降解研究的方向提出了展望。     

模拟微重力条件下大鼠肝细胞三维培养支架材料的筛选（简报）

组织工程是一门新兴的边缘学科,它是利用体外培养的人体功能细胞与适当的细胞外基质或支架材料相结合,然后将其移植到体内病损部位以期达到修复目的。微重力组织工程(Microgravity Tissue Engineering)是近年来由美国空间生物技术研究人员开创的一个独特研究领域,其核心技术是     

模拟微重力条件下大鼠肝细胞三维培养支架材料的筛选(简报)

组织工程是一门新兴的边缘学科,它是利用体外培养的人体功能细胞与适当的细胞外基质或支架材料相结合,然后将其移植到体内病损部位以期达到修复目的。微重力组织工程(Microgravity Tis-sue Engineering)是近年来由美国空间生物技术研究人员开创的一个独特研究领域,其核心技术是建立微重力条件下哺乳动物细胞三维(Three Dimen-sion)培养体系。利用外壁转动生物反应器(RotatingWall Vessel Bioreactor,RWVB)模拟微重力培养环境,减少培养液对细胞产生的机械剪切力,增加细胞营养的补充,加速代谢产物的排除,因此可以大大改善离体细胞的培养条件,使在普通重力培养条件下只能二维贴壁生长的哺乳动物细胞表现出三维增殖与分化,这类分化的细胞团可进一步形成有功能的     

Degradability of polysaccharides multilayer films in the oral environment: an in vitro and in vivo study

Biomedical devices and modified biomaterial surfaces constitute an expanding research domain in the dental field. However, such oral applications have to face a very particular environment containing specific physiological conditions and specific enzymes. To evaluate their suitability in the development of novel oral applications, the degradability of polyelectrolyte multilayer films made of the natural polysaccharides chitosan and hyaluronan (CHI/HA) was investigated in vitro and in vivo in a rat mouth model. The films were either native or cross-linked using a water-soluble carbodiimide (EDC) in combination with N-hydroxysulfosuccinimide. The in vitro degradation of the films by different enzymes present in the oral environment, such as lysozyme and amylase, was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy observations after being film labeled with CHI(FITC). Whereas native films were subjected to degradation by all the enzymes, cross-linked films were more resistant to enzymatic degradation. Films were also put in contact with whole saliva, which induced a slow degradation of the native films over an 18 h period. The in vivo degradation of the films deposited on polymer disks and sutured in the rat mouth was followed over a 3 day period. Whereas film degradation is fast for native films, it is much slower for the cross-linked ones. More than 60% of these films remained on the disks after 3 days in the mouth. Taken together, these results suggest that the multilayer films made of natural polysaccharides are of high potential interest for oral applications, especially as drug release systems, offering various degradation rates and consequent release characteristics.     

Structure and morphology changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber membrane

Electrospun poly(glycolide-co-lactide) (PLA10GA90, LA/GA ratio 10/90) biodegradable nanofiber membranes possessed very high surface area to volume ratios and were completely noncrystalline with a relatively lowered glass transition temperature. These characteristics led to very different structure, morphology, and property changes during in vitro degradation, which were examined systematically. A shrinkage study showed that the electrospun crystallizable but amorphous PLA10GA90 membranes exhibited a very small shrinkage percentage when compared with the electrospun membranes of noncrystallizable poly(lactide-co-glycolide) (PLA75GA25, LA/GA 75/25) and poly(d,l-lactide). Although the weight loss of electrospun PLA10GA90 membranes exhibited a similar degradation behavior as cast thin films, detailed studies showed that the structure and morphology changes in electrospun membranes followed different pathways during the hydrolytic degradation. After 1 day of degradation in buffer solution at 37 degrees C, electrospun PLA10GA90 membranes exhibited a sudden increase in crystallinity and glass transition temperature, due to the fast thermally induced crystallization process. The continuous increase in crystallinity and apparent crystal size, as well as the decrease in long period and lamellae thickness, indicated that the thermally induced crystallization was followed by a chain cleavage induced crystallization process. The mass loss rate was accelerated after 6 days of degradation. The increase in glass transition temperature during this period further confirmed that the degradation of PLA10GA90 nanofibers was initiated from the amorphous region within the lamellar superstructures. A mechanism of structure and morphology changes during in vitro degradation of electrospun PLA10GA90 nanofibers is proposed.     

Biocompatibility and long-term toxicity of InnoPol implant, a biodegradable polymer scaffold.

InnoPol, a poly((D,L)-lactic-co-glycolic acid) [PLGA] 65/35 scaffold manufactured by special gas foaming methods in Korea, was subjected to tests to evaluate the degradation and tissue compatibility characteristics and long-term systemic toxicity in mice and rats. C57BL/6 mice and SD rats were implanted subcutaneously with 3-mm- and 1-mm-thick InnoPol circular discs, 10 mm in diameter, respectively, and sacrificed 8, 12, and 24 weeks after implantation. No test material-related effects were observed in mortality, clinical signs, body weight gain, food and water consumption, ophthalmologic signs, urinalysis, hematology, serum biochemistry parameters and organ weights of all animals implanted with InnoPol. Also, there were no systemic symptoms including metabolic alterations and inflammatory reactions in either mice or rats. In addition, no gross pathological findings, except skin lesions around the implantation sites, were found in the major organs. Although mild inflammation at the site of InnoPol implantation was confirmed from hematoxylin and eosin or Masson's trichrome staining at 8-12 weeks, the reactions had disappeared at 24 weeks following complete degradation of the scaffold, leaving granulomatous tissues that were similar to surgical wounds in sham operation controls without implants. These results suggest that InnoPol possesses good mechanical properties and tissue compatibility and does not cause any systemic toxicity other than transient local inflammatory reactions at the implantation site, and that it might be useful in applications as a medical device for implantation.     

Poly(L-lactide-co-glycolide) thin films can act as autologous cell carriers for skin tissue engineering

Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.     

Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts

Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60-88 μm), type II (88-100 μm) and type III (100-125 μm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague-Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.     

Nanofibrous scaffolds incorporating PDGF-BB microspheres induce chemokine expression and tissue neogenesis in vivo

Platelet-derived growth factor (PDGF) exerts multiple cellular effects that stimulate wound repair in multiple tissues. However, a major obstacle for its successful clinical application is the delivery system, which ultimately controls the in vivo release rate of PDGF. Polylactic-co-glycolic acid (PLGA) microspheres (MS) in nanofibrous scaffolds (NFS) have been shown to control the release of rhPDGF-BB in vitro. In order to investigate the effects of rhPDGF-BB release from MS in NFS on gene expression and enhancement of soft tissue engineering, rhPDGF-BB was incorporated into differing molecular weight (MW) polymeric MS. By controlling the MW of the MS over a range of 6.5 KDa-64 KDa, release rates of PDGF can be regulated over periods of weeks to months in vitro. The NFS-MS scaffolds were divided into multiple groups based on MS release characteristics and PDGF concentration ranging from 2.5-25.0 microg and evaluated in vivo in a soft tissue wound repair model in the dorsa of rats. At 3, 7, 14 and 21 days post-implantation, the scaffold implants were harvested followed by assessments of cell penetration, vasculogenesis and tissue neogenesis. Gene expression profiles using cDNA microarrays were performed on the PDGF-releasing NFS. The percentage of tissue invasion into MS-containing NFS at 7 days was higher in the PDGF groups when compared to controls. Blood vessel number in the HMW groups containing either 2.5 or 25 microg PDGF was increased above those of other groups at 7d (p<0.01). Results from cDNA array showed that PDGF strongly enhanced in vivo gene expression of the CXC chemokine family members such as CXCL1, CXCL2 and CXCL5. Thus, sustained release of rhPDGF-BB, controlled by slow-releasing MS associated with the NFS delivery system, enhanced cell migration and angiogenesis in vivo, and may be related to an induced expression of chemokine-related genes. This approach offers a technology to accurately control growth factor release to promote soft tissue engineering in vivo.     

Biomaterials

Biomaterials--materials used for the elaboration of systems designed for human implantation or organ substitutes--can be classified as metals and alloys, ceramics and polymers. Their uses are largely diversified, for soft and hard tissues replacement. Interactions rise between biological environment and implants, the mechanisms of them not always known: inflammatory response, corrosion and degradation of materials leading to leaching of some constituents possibly toxic and alteration of their mechanical properties. Blood interfacing materials introduce some particular problems of hemocompatibility. The matching of implant to biological medium, in other words, its biocompatibility has to be a priori evaluated, but until now no in vitro or in vivo evaluation method is fully reliable.     

PGLA编织支架的制备与初步体内评价

目的:热拉伸会改变纤维的结构和性能,进而影响由纤维编织而成的支架的性能。本文考察了PGLA纤维的拉伸倍数对编织支架在SD大鼠皮下的体内降解行为的影响。方法:制备了基于生物可降解高分子材料聚乙交酯丙交酯(PGLA,GA/LA摩尔比=90/10)的完全生物可降解编织支架,通过测试支架在大鼠体内降解过程中的失重、表面形貌、热性能、径向压缩力等变化情况,考察了纤维的不同的拉伸倍数对支架体内降解过程的影响。结果:用拉伸倍数为5的PGLA纤维编织的支架在植入SD大鼠皮下后降解最慢,重量、吸水率、结晶度、化学成分和径向压缩力的变化最慢,植入体内10天后能够保持完整的支架形态。结论:纤维的拉伸倍数会影响由纤维编织成的支架的热性能和力学性能的变化,本研究结果表明这种新的手工编织的支架具有短暂支撑管腔狭窄的潜在应用,为支架的材料选择和制备方法提供了参考,为在体内起到短暂支撑作用的支架的深入研究提供了实验基础。     

In vivo degradation behavior of photo-cross-linked star-poly(epsilon-caprolactone-co-D,L-lactide) elastomers

We have recently reported on the preparation of biodegradable elastomers through photo-cross-linking acrylated star-poly(epsilon-caprolactone-co-D,L-lactide). In this paper we assess the change in their physical properties during in vivo degradation in rats after subcutaneous implantation over a 12 week period. These parameter changes were compared to those observed in vitro. Two different cross-link densities were examined, representing the range from a high Young's modulus to a low Young modulus. Elastomers having a high cross-link density exhibited degradation behavior consistent with a surface erosion mechanism, and degraded at the same rate in vivo as observed in vitro. Young's modulus and the stress at break of these elastomers decreased linearly with the degradation time, while the strain at break decreased slowly. Elastomers having a low cross-link density exhibited a degradation mechanism consistent with bulk erosion. Young's modulus and the stress at break of these elastomers decreased slowly initially, followed by a marked increase in mechanical strength loss after 4 weeks. The elastomers were well tolerated by the rats over the 12 week period in vivo.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

Formation of acylated growth hormone-releasing peptide-6 by poly(lactide-co-glycolide) and its biological activity

The purpose of this study was to investigate the formation of acylated impurity resulting from a chemical reaction between the growth hormone-releasing peptide-6 (GHRP-6) and poly(lactide-co-glycolide) (PLGA) and the effect of peptide acylation on the in vivo biological activity of GHRP-6. The peptide acylation pattern of GHRP-6 by hydrophilic PLGA polymers with different molecular weights was characterized by reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Higher levels of acylated GHRP-6 were produced with the higher molecular weight PLGA, which might be due to the slower degradation rate of the polymer. The evaluation of the biological activity in rats showed that the acylated GHRP-6 had a much lower activity than the intact GHRP-6. This finding suggests that the acylation reaction would decrease the effectiveness of the GHRP-6 formulation such as PLGA microspheres. There-fore, a strategy for stabilizing the GHRP-6 will be necessary for the development of a successful formulation of PLGA microspheres. Published: June 8, 2007     

The pro‐inflammatory response of macrophages regulated by acid degradation products of poly(lactide‐co‐glycolide) nanoparticles

Poly(lactide‐co‐glycolide) (PLGA) shows great potentials in biomedical applications, in particular with the field of biodegradable implants and control release technologies. However, there are few systematic and detailed studies on the influence of PLGA degradation behavior on the immunogenicity. In this study, in order to develop a method for dynamically assessing the immunological response of PLGA throughout the implantation process, PLGA particles are fabricated using an o/w single‐emulsion method. The physicochemical characterizations of the prepared PLGA particles during in vitro hydrolytic degradation are investigated. Then, a series of immunological effects triggered by PLGA by‐products formed with degradation process are evaluated, including cell viability, apoptosis, polarization and inflammatory reaction. THP‐1 human cell line is set as in vitro cell model. Our results show that PLGA degradation‐induced acid environment decreases cell viability and increases cell apoptosis, which is a potential factor affecting cell function. In particular, the macrophages exhibit up‐regulations in both M1 subtype related surface markers and pro‐inflammatory cytokines with the degradation process of PLGA, which indicates the degradation products of PLGA can convert macrophages to the pro‐inflammatory (M1) polarization state. All these findings provide the mechanism of PLGA‐induced inflammation and lay the foundation for the design of next‐generation PLGA‐based biomaterials endowed with immunomodulatory functions.     

Versatility of biodegradable biopolymers: degradability and an in vivo application

Biodegradable materials have various important applications in the biomedical field. There are basically two groups of polyesters which have significant importance in this field. These are polylactides and polyhydroxybutyrates. Both groups degrade via hydrolysis with the rates of degradation depending on medium properties such as pH, temperature, solvent and presence of biocatalysts, as well as on chemical compositions. In order for these biomaterials to be suitable for use in load bearing applications without deformation or warping their strengths and their capability to maintain their form must be improved. To insure dimensional stability during degradation and to match modulus and strength to that of bone, introduction of a reinforcing structure for those applications to plate fixation through the creation of an interpenetrating network might be a feasible approach. In this study, poly(lactide-co-glycolide) (PLGA), was the major structural element to be strengthened by a three-dimensional network or "scaffold" of another biodegradable polymer, poly(propylene fumarate) (PPF). PPF would be crosslinked with a biocompatible vinyl monomer, vinylpyrrolidone (VP). Three different approaches were tested to create dimensionally stable bone plates. First, via in situ crosslinking of PPF in the presence of PLGA. Secondly, by blending of precrosslinked PPF with PLGA. Finally, by simultaneous crosslinking and molding of the PLGA, PPF and VP. These were compared against extruded or compression molded PLGA controls. Results showed that compression molding at room temperature followed by crosslinking under pressure at elevated temperature and subsequently by gamma-irradiation appeared to yield the most favorable product as judged by swelling, hardness and flexural strength data. The composition of the implant material, PLGA(3):PPF(1):VP(0.7), appeared to be suitable and formed the compositional and procedural basis for in vivo biocompatibility studies.     

Biocompatibility studies of modified collagen/Hyaluronan membranes after implantation

Two varieties of collagen/sodium hyaluronan membranes were used asdermal substitutes in a biocompatibility implantation study on rats. In ordertoimprove especially the physical and mechanical properties of the material, themembranes were chemically modified using a combination ofhexamethylenediisocyanate (HMDC) as a crosslinker and polyoxy-ethylene (POE) asa spacer. According to both macroscopic and microscopic histologicalobservations, the membranes were well accepted by the surrounding host tissueinall the animals. No major differences in relation to the outgrowth of thematerial by host tissue have been observed between the implant varieties A andB. The most important finding was that no pathological changes or importantalterations of the host tissues were detected.     

Biodegradable Monocrystalline Silicon Photovoltaic Microcells as Power Supplies for Transient Biomedical Implants

Bioresorbable electronic materials serve as foundations for implantable devices that provide active diagnostic or therapeutic function over a timeframe matched to a biological process, and then disappear within the body to avoid secondary surgical extraction. Approaches to power supply in these physically transient systems are critically important. This paper describes a fully biodegradable, monocrystalline silicon photovoltaic (PV) platform based on microscale cells (microcells) designed to operate at wavelengths with long penetration depths in biological tissues (red and near infrared wavelengths), such that external illumination can provide realistic levels of power. Systematic characterization and theoretical simulations of operation under porcine skin and fat establish a foundational understanding of these systems and their scalability. In vivo studies of a representative platform capable of generating ≈60 µW of electrical power under 4 mm of porcine skin and fat illustrate an ability to operate blue light‐emitting diodes (LEDs) as subdermal implants in rats for 3 d. Here, the PV system fully resorbs after 4 months. Histological analysis reveals that the degradation process introduces no inflammatory responses in the surrounding tissues. The results suggest the potential for using silicon photovoltaic microcells as bioresorbable power supplies for various transient biomedical implants.