壳聚糖大鼠颅内组织相容性的初步观察

本实验探讨了生物可降解的天然高分子缓释材料壳聚糖在脑组织中的生物相容性.以期为临床提供可用于颅内植入化疗的药物缓释高分子载体.我们以32只SD大鼠为研究对象,随机分为实验组和对照组,分别植入壳聚糖和明胶海绵.观察术后的行为改变和3,7,14,30天时的局部组织反应(HE染色).观察发现所有动物无明显行为改变,术后3,7,14,30天的组织学观察表明壳聚糖与明胶海绵有类似的异物反应.因此壳聚糖具有良好的脑组织生物相容性,并可安全的降解.可作为颅内植入缓释化疗药物的载体.     

The simultaneous effect of polymer chemistry and device geometry on the in vitro activation of murine dendritic cells

Polyanhydrides are a promising class of biomaterials for use as vaccine adjuvants and as multi-component implants. Their properties can be tailored for such applications as controlled drug release, drug stability, and/or immune regulation (adjuvant effect). Understanding the induction of immunomodulatory mechanisms of this polymer system is important for the design and development of efficacious vaccines and tissue compatible multi-component implantable devices using this polymer system. This study describes the development of a rapid multiplexed method for the investigation of the adjuvanticity of polyanhydride nanospheres and films using murine dendritic cells (DCs). To assess the immune response, cell surface markers including MHC II, CD86, CD40, and CD209 and cytokines including IL-6, IL-12p40, and IL-10 were measured. The DCs incubated with nanospheres displayed enhanced expression of all the surface markers and the production of IL-12p40 compared to DCs incubated with polymer films in a chemistry-dependent manner. This suggests that polyanhydrides of various chemistries and device geometries can be tailored to achieve desired levels of immune cell activation for specific applications. The observed biocompatibility and activation of DCs by polyanhydride devices supports their inclusion in vaccine delivery devices as well as in multi-component medical implants.     

Polyanhydride microparticles enhance dendritic cell antigen presentation and activation

The present study was designed to evaluate the adjuvant activity of polyanhydride microparticles prepared in the absence of additional stabilizers, excipients or immune modulators. Microparticles composed of varying ratios of either 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid or 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and CPH were added to in vitro cultures of bone marrow-derived dendritic cells (DCs). Microparticles were efficiently and rapidly phagocytosed by DCs in the absence of opsonization and without centrifugation or agitation. Within 2h, internalized particles were rapidly localized to an acidic, phagolysosomal compartment. By 48 h, only a minor reduction in microparticle size was observed in the phagolysosomal compartment, indicating minimal particle erosion consistent with being localized within an intracellular microenvironment favoring particle stability. Polyanhydride microparticles increased DC surface expression of major histocompatability complex class II, the co-stimulatory molecules CD86 and CD40, and the C-type lectin CIRE (murine DC-SIGN; CD209). In addition, microparticle stimulation of DCs also enhanced secretion of the cytokines IL-12p40 and IL-6, a phenomenon found to be dependent on polymer chemistry. DCs cultured with polyanhydride microparticles and ovalbumin induced polymer chemistry-dependent antigen-specific proliferation of both CD4(+) OT-II and CD8(+) OT-I T cells. These data indicate that polyanhydride particles can be tailored to take advantage of the potential plasticity of the immune response, resulting in the ability to induce immune protection against many types of pathogens.     

Design and evaluation of novel polyanhydride blends as nerve guidance conduits

Implantable biodegradable nerve guidance conduits (NGCs) have the potential to align and support regenerating cells, as well as prevent scar formation. In this study in vitro bioassays and in vivo material evaluations were performed using a nerve guidance conduit material made from a novel polyanhydride blend. In vitro cytotoxicity studies with both fibroblasts and primary chick neurons demonstrated that the proposed polyanhydride blend was non-cytotoxic. Subcutaneous implantation for 7 days in rats resulted in an initial fibrin matrix, minimal macrophage presence and angiogenesis in the surrounding tissues. Nerve guidance conduits fabricated from the proposed polyanhydride blend material may serve as favorable biocompatible tissue engineering devices.     

In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: direct contact studies

Synthetic biodegradable polymers have many potential therapeutic applications. In ophthalmology, biodegradable polymers have been used as viscoelastic agents and surgical implants. Other potential applications include controlled release of drugs and growth factors, gene therapy, and tissue engineering. In the present study, in vitro biocompatibility of three biodegradable polymers, 50:50 PDLGA, 85:15 PDLGA, and Inion GTR membrane was evaluated in comparison to tissue culture polystyrene by investigating cell proliferation and potential acute toxicity by the WST-1 cytotoxicity/cell proliferation test, the ATP test, and the lactate dehydrogenase (LDH) test. Evaluations were conducted with cell line cultures from various ocular tissues, human corneal epithelial cells (HCE), rabbit stromal fibroblasts (SIRC), bovine corneal endothelial cells (BCE), human conjunctival epithelial cells (IOBA-NHC), and human retinal pigment epithelial cells (ARPE-19) by direct contact studies by plating the cells on the polymer film specimens in 96-wells. The proliferation results show that cell lines from various ocular tissues attached and grew on PDLGA 50:50, PDLGA 85:15, and Inion GTR membrane. Cytotoxicity experiments with the LDH and ATP tests showed no or extremely slight toxic adverse effects. These polymers have potential to be used as scaffolds in cell transplantation devices or as surgical implants.     

Curcumin-releasing mechanically adaptive intracortical implants improve the proximal neuronal density and blood–brain barrier stability

The cellular and molecular mechanisms by which neuroinflammatory pathways respond to and propagate the reactive tissue response to intracortical microelectrodes remain active areas of research. We previously demonstrated that both the mechanical mismatch between rigid implants and the much softer brain tissue, as well as oxidative stress, contribute to the neurodegenerative reactive tissue response to intracortical implants. In this study, we utilize physiologically responsive, mechanically adaptive polymer implants based on poly(vinyl alcohol) (PVA), with the capability to also locally administer the antioxidant curcumin. The goal of this study is to investigate if the combination of two independently effective mechanisms – softening of the implant and antioxidant release – leads to synergistic effects in vivo. Over the first 4 weeks of the implantation, curcumin-releasing, mechanically adaptive implants were associated with higher neuron survival and a more stable blood–brain barrier at the implant–tissue interface than the neat PVA controls. 12 weeks post-implantation, the benefits of the curcumin release were lost, and both sets of compliant materials (with and without curcumin) had no statistically significant differences in neuronal density distribution profiles. Overall, however, the curcumin-releasing softening polymer implants cause minimal implant-mediated neuroinflammation, and embody the new concept of localized drug delivery from mechanically adaptive intracortical implants.     

Composite resorbable polymer/hydroxylapatite composite screws for fixation of osteochondral osteotomies

The aim of this study was to evaluate biomechanically the healing of an osteochondral fragment created in the distal sheep femur in response to fixation with a resorbable composite screw made of polylactide and hydroxylapatite. Pure poly(L-lactide) screws were used for comparison. At follow-up times of 4 or 8 weeks, specimens were examined with standard radiography, biomechanics, and histology. The intact contralateral femur served as a control. Only minimal signs of polymer degradation were seen in the histologic specimens. At 8 weeks, most osteotomies had healed completely and there was no difference in compressive strength and elastic modulus of cylindrical cores between the two types of biodegradable implants used. The width of the repair tissue at the tissue-implant interface was 250+/-50 micro m representing a clear transition zone of newly formed trabecular bone separating the implant from the surrounding plexiform bone. We conclude that relatively large polylactide implants, blended with hydroxyapatite, are capable of fixing an osteochondral fragment in an animal model. Biomechanical data assessing the quality of the bone formed at the osteotomy sites were found to be equivalent when compared to the control poly(L-lactide) implants of similar design and size. In addition, hydroxylapatite composite implants showed benign tissue responses and good implant osteointegration. Results suggest that hydroxylapatite composite screw implants can be used for similar indications as pure poly(L-lactide) implants in current clinical use.     

Brain tissue reaction to permselective polymer capsules

The brain tissue reaction to permselective polymer capsules implanted in rats was evaluated for 1 to 54 weeks. The polymer capsules were well tolerated in all animals and no recognizable neurological or behavioral deficits were associated with the implants. Necrosis at the brain/polymer interface, as assessed with Nissl stain, was not observed. Foreign body giant cells were consistently absent. Immunocytochemically identified reactive neuroglial cells showed a remarkably low-grade tissue response to the synthetic material beyond the first 2 weeks of observation. Immunolabeled cortical neurons revealed conserved columnar arrays around the implants. Transmission electron microscopy showed a minimal degree of collagen deposition compared to implants in peripheral sites, and normal synapses within a few micrometers from the brain/polymer interface, supporting the prospect of biocompatible, immunoisolated xenografts in the central nervous system.     

Degradable poly(anhydride ester) implants: effects of localized salicylic acid release on bone

Degradable poly(anhydride ester) implants in which the polymer backbone breaks down into salicylic acid (SA) were investigated. In this preliminary work, local release of SA from the poly(anhydride esters), thus classified as ‘active polymers', on healthy bone and tissue was evaluated in vivo using a mouse model. Degradable polyanhydrides that break down into inactive by-products were used as control membranes because of their chemical similarity to the active polymers. Small polymer squares were inserted over the exposed palatal bone adjacent to the maxillary first molars. Active polymer membranes were placed on one side of the mouth, control polymers placed on the contra lateral side. Intraoral clinical examination showed that active polymer sites were less swollen and inflamed than control polymer sites. Histopathological examination at day 1 showed essentially no difference between control and active polymers. After 4 days, active polymer sites showed epithelial proliferation to a greater extent than the polyanhydride controls. After 20 days, active polymer sites showed greater thickness of new palatal bone and no resorptive areas, while control polymer sites showed less bone thickness as well as resorption including lacunae involving cementum and dentine. From these preliminary studies, we conclude that active polymers, namely poly(anhydride esters), stimulated new bone formation.     

Single dose vaccine based on biodegradable polyanhydride microspheres can modulate immune response mechanism

This study focuses on the development of single dose vaccines based on biodegradable polyanhydride microspheres that have the unique capability to modulate the immune response mechanism. The polymer system employed consists of copolymers of 1,6-bis(p-carboxyphenoxy)hexane and sebacic acid. Two copolymer formulations that have been shown to provide extended release kinetics and protein stability were investigated. Using tetanus toxoid (TT) as a model antigen, in vivo studies in C3H/HeOuJ mice demonstrated that the encapsulation procedure preserves the immunogenicity of the TT. The polymer itself exhibited an adjuvant effect, enhancing the immune response to a small dose of TT. The microspheres provided a prolonged exposure to TT sufficient to induce both a primary and a secondary immune response (i.e., high antibody titers) with high-avidity antibody production, without requiring an additional administration. Antigen-specific proliferation 28 weeks after a single immunization indicated that immunization with the polyanhydride microspheres generated long-lived memory cells and plasma cells (antibody-secreting B cells) that generally do not occur without maturation signals from T helper cells. Furthermore, by altering the vaccine formulation, the overall strength of the T helper type 2 immune response was selectively diminished, resulting in a balanced immune response, without reducing the overall titer. This result is striking, considering free TT induces a T helper type 2 immune response, and has important implications for developing vaccines to intracellular pathogens. The ability to selectively tune the immune response without the administration of additional cytokines or noxious adjuvants is a unique feature of this delivery vehicle that may make it an excellent candidate for vaccine development.     

Combined radiofrequency ablation and doxorubicin-eluting polymer implants for liver cancer treatment

Previously, biodegradable polymer implants (polymer millirods) to release chemotherapeutic agents directly into tumors have been developed. The purpose of this study is to evaluate local drug distribution from these implants in liver tumors treated with radiofrequency (RF) ablation and determine if the implants provide a therapeutic improvement over RF ablation alone. Cylindrical implants were fabricated using 65% poly(D,L-lactide-co-glycolide) (PLGA), 21.5% NaCl, and 13.5% doxorubicin. Control or drug-containing millirods were implanted inside VX2 liver tumors (11 mm diameter) in rabbits after RF ablation. Therapeutic efficacy was assessed 4 and 8 days after treatment using tumor size, histology, and fluorescence measurement of drug distribution. Tumors in both test groups recurred at the boundary of the ablated region. Therapeutic doxorubicin concentrations were found in more than 80% of the ablated area, but concentrations declined rapidly at the boundary between normal and ablated tissue. This region was characterized by a developing fibrous capsule with resolving inflammation, which restricted drug transport out of the ablated zone. The intratumoral doxorubicin implants delivered high concentrations of drug within the ablated region but only limited amounts outside the ablation zone. Future studies will focus on overcoming the fibrotic transport barrier and enhancing drug delivery to the periphery of the ablation region to prevent tumor progression.     

Multiple forms of monoamine oxidase in human brain tumors

The multiplicity of monoamine oxidase (MAO) in human brain tumors was studied with a criterion of inhibitor sensitivity. All of the human brain tumors of glial origin, irrespective of their malignances, showed similar or lower ratios of type A MAO to type B MAO as compared with the normal cerebral hemispheres. This result is quite different from those of mouse neuroblastoma and rat glioma.     

A unified mathematical model for the prediction of controlled release from surface and bulk eroding polymer matrices

A unified model has been developed to predict release not only from bulk eroding and surface eroding systems but also from matrices that transition from surface eroding to bulk eroding behavior during the course of degradation. This broad applicability is afforded by fundamental diffusion/reaction equations that can describe a wide variety of scenarios including hydration of and mass loss from a hydrolysable polymer matrix. Together, these equations naturally account for spatial distributions of polymer degradation rate. In this model paradigm, the theoretical minimal size required for a matrix to exhibit degradation under surface eroding conditions was calculated for various polymer types and then verified by empirical data from the literature. An additional set of equations accounts for dissolution- and/or degradation-based release, which are dependent upon hydration of the matrix and erosion of the polymer. To test the model's accuracy, predictions for agent egress were compared to experimental data from polyanhydride and polyorthoester implants that were postulated to undergo either dissolution-limited or degradation-controlled release. Because these predictions are calculated solely from readily attainable design parameters, it seems likely that this model could be used to guide the design controlled release formulations that produce a broad array of custom release profiles.     

Intracranial microcapsule drug delivery device for the treatment of an experimental gliosarcoma model

Controlled-release drug delivery systems are capable of treating debilitating diseases, including cancer. Brain cancer, in particular glioblastoma multiforme (GBM), is an extremely invasive cancer with a dismal prognosis. The use of drugs capable of crossing the blood-brain barrier has shown modest prolongation in patient survival, but not without unsatisfactory systemic, dose-limiting toxicity. Among the reasons for this improvement include a better understanding of the challenges of delivery of effective agents directly to the brain tumor site. The combination of carmustine delivered by biodegradable polyanhydride wafers (Gliadel(?)), with the systemic alkylating agent, temozolomide, allows much higher effective doses of the drug while minimizing the systemic toxicity. We have previously shown that locally delivering these two drugs leads to further improvement in survival in experimental models. We postulated that microcapsule devices capable of releasing temozolomide would increase the therapeutic capability of this approach. A biocompatible drug delivery microcapsule device for the intracranial delivery of temozolomide is described. Drug release profiles from these microcapsules can be modulated based on the physical chemistry of the drug and the dimensions of the release orifices in these devices. The drug released from the microcapsules in these experiments was the clinically utilized chemotherapeutic agent, temozolomide. In vitro studies were performed in order to test the function, reliability, and drug release kinetics of the devices. The efficacy of the temozolomide-filled microcapsules was tested in an intracranial experimental rodent gliosarcoma model. Immunohistochemical analysis of tissue for evidence of DNA strand breaks via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. The experimental release curves showed mass flow rates of 36?μg/h for single-orifice devices and an 88?μg/h mass flow rate for multiple-orifice devices loaded with temozolomide. In vivo efficacy results showed that localized intracranial delivery of temozolomide from microcapsule devices was capable of prolonging animal survival and may offer a novel form of treatment for brain tumors.     

Polymers to treat brain tumours

Brain tumours are difficult to treat by conventional methods. A biodegradable polymer, poly-[bis(p-carboxyphenoxy) propane sebacic acid] with a nitrosourea, carmustine, has been demonstrated to be biocompatible in the brains of experimental animals and to release drugs in a predictable sustained manner. Carmustine impregnated in polymers appears to be more effective than when delivered by standard methods. A Phase I clinical study has demonstrated the safety of this approach in treating brain tumours and a Phase III placebo-controlled study is currently underway. Other applications of the polymer in the treatment of brain diseases are discussed.     

Fabrication and biocompatibility of polypyrrole implants suitable for neural prosthetics

Finding a conductive substrate that promotes neural interactions is an essential step for advancing neural interfaces. The biocompatibility and conductive properties of polypyrrole (PPy) make it an attractive substrate for neural scaffolds, electrodes, and devices. Stand-alone polymer implants also provide the additional advantages of flexibility and biodegradability. To examine PPy biocompatibility, dissociated primary cerebral cortical cells were cultured on PPy samples that had been doped with polystyrene-sulfonate (PSS) or sodium dodecylbenzenesulfonate (NaDBS). Various conditions were used for electrodeposition to produce different surface properties. Neural networks grew on all of the PPy surfaces. PPy implants, consisting of the same dopants and conditions, were surgically implanted in the cerebral cortex of the rat. The results were compared to stab wounds and Teflon implants of the same size. Quantification of the intensity and extent of gliosis at 3- and 6-week time points demonstrated that all versions of PPy were at least as biocompatible as Teflon and in fact performed better in most cases. In all of the PPy implant cases, neurons and glial cells enveloped the implant. In several cases, neural tissue was present in the lumen of the implants, allowing contact of the brain parenchyma through the implants.     

骨植入用生物可降解材料新合成方法及生物相容性的研究

采用 Y(CF3COO) 3/AL(i- Bu) 3络合物催化合成聚己内酯 (Polycaprolactone,PCL)及其与聚乳酸(Polylactic acid,PL A)的共聚物 ,并应用生物化学、细胞毒理及细胞免疫学等实验方法对这一新型骨科生物降解可吸收材料的生物相容性进行评价。用 PCL 和 PCL/PDL L A共聚物作为骨科生物降解可吸收内置物材料 ,国内外属首次研究 ;多聚物的分子量和降解时间可以通过控制反应时间、单体与催化剂的比例来调控产物分子量 ,人为调节降解时间。实验证明该材料细胞相容性好 ,未发现明显毒性及免疫排斥反应。     

A new bone-inducing biodegradable porous beta-tricalcium phosphate

A new type of degradable biomaterial with bone-inducing capacity was made by combining porous beta-tricalcium phosphate (beta-TCP) with a delivery system for recombinant human bone morphogenetic protein-2 (rhBMP-2). The BMP delivery system consisted of a block copolymer composed of poly-D,L-lactic acid with random insertion of p-dioxanone and polyethylene glycol (PLA-DX-PEG), a known biocompatible and biodegradable material. The efficacy of this biomaterial in terms of its bone-inducing capacity was examined by ectopic bone formation in the dorsal muscles of the mouse. In the beta-TCP implants coated with the PLA-DX-PEG polymer containing more than 0.0025% (w/w) of rhBMP-2, new ectopic bone tissues with marrow were consistently found on the surface of implants. The radiographic density of beta-TCP was diminished in a time-dependent manner. On histological examination, numerous multinucleated osteoclasts with positive tartrate-resistant acid-phosphatase (TRAP) staining were noted on the surface of the beta-TCP. These experimental results indicate that beta-TCP implants coated with synthetic rhBMP-2 delivery system might provide effective artificial bone-graft substitutes with osteoinductive capacity and biodegradable properties. In addition, this type of biomaterial may require less rhBMP-2 to induce significant new bone mass.     

Short term in vivo biocompatibility testing of biodegradable poly(D,L-lactide)--growth factor coating for orthopaedic implants

Fracture healing can be stimulated by exogenous application of growth factors. Using porcine and rat models the efficacy of locally delivered IGF-I and TGF-beta1 from an implant coating has been demonstrated. A thin and biomechanical stable biodegradable poly(D,L-lactide) was used to coat implants and serve as a drug carrier. Due to reports of possible foreign body reactions caused by polymer materials in orthopedic surgery, this study investigated the biocompatibility of the polylactide implant coating and the locally released growth factors during the time course of rat tibial fracture healing (days 5, 10, 15, and 28 after fracture). Monocytes/macrophages and osteoclast were detected using an monoclonal antibody against ED1 (comparable to CD68 in mice and human). The antibody ED1 stains monocytes, macrophages and osteoclast in the bone marrow and in the newly formed fracture callus. A moderate density of the monocytes/macrophages was seen in the proximal part of the medullary canal, but almost no cells were detectable in the region distal to the fracture. The amount of stained cells increased during the observation time with a maximum at days 10 and 15 followed by a decrease at day 28. No differences were detectable between the investigated groups from day 5 to 15 post fracture indicating, that the used poly(D,L-lactide) or the incorporated growth factors do not evoke an elevated immunological response compared to the uncoated titanium implant at the investigated time points. A significantly higher amount of ED1 positive cells was measured 28 days after fracture in the control group compared to the groups with the coated implants. In conclusion, no indication of a foreign body reaction due to the use of the polylactide or the growth factors was found indicating a good short-term biocompatibility of this bioactive coating.     

Vascularization and tissue infiltration of a biodegradable polyurethane matrix

Urethanes are frequently used in biomedical applications because of their excellent biocompatibility. However, their use has been limited to bioresistant polyurethanes. The aim of this study was to develop a nontoxic biodegradable polyurethane and to test its potential for tissue compatibility. A matrix was synthesized with pentane diisocyanate (PDI) as a hard segment and sucrose as a hydroxyl group donor to obtain a microtextured spongy urethane matrix. The matrix was biodegradable in an aqueous solution at 37 degrees C in vitro as well as in vivo. The polymer was mechanically stable at body temperatures and exhibited a glass transition temperature (Tg) of 67 degrees C. The porosity of the polymer network was between 10 and 2000 microm, with the majority of pores between 100 and 300 microm in diameter. This porosity was found to be adequate to support the adherence and proliferation of bone-marrow stromal cells (BMSC) and chondrocytes in vitro. The degradation products of the polymer were nontoxic to cells in vitro. Subdermal implants of the PDI-sucrose matrix did not exhibit toxicity in vivo and did not induce an acute inflammatory response in the host. However, some foreign-body giant cells did accumulate around the polymer and in its pores, suggesting its degradation is facilitated by hydrolysis as well as by giant cells. More important, subdermal implants of the polymer allowed marked infiltration of vascular and connective tissue, suggesting the free flow of fluids and nutrients in the implants. Because of the flexibility of the mechanical strength that can be obtained in urethanes and because of the ease with which a porous microtexture can be achieved, this matrix may be useful in many tissue-engineering applications.