The chick chorioallantoic membrane as a novel in vivo model for the testing of biomaterials

Current in vivo models for testing biomaterials are time and labor intensive as well as expensive. This article describes a new approach for testing biomaterials in vivo using the chorioallantoic membrane (CAM) of the developing chicken embryo, as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 4 days, at which time a small window was cut in the shell of the egg. After 1 week of incubation, the CAM received several test materials, including the endotoxin LPS, a cotton thread and a Silastic tubing. One day and 1 week later, the tissue response to the test materials was assessed using gross, histological, and scanning electron microscope evaluations. The inflammatory response of the chorioallantoic membrane to biomaterials was fully characterized and found to be similar to that of the mammalian response and was also seen to vary according to test materials. We also found that the structure and geometry of the test materials greatly influenced the incorporation of the samples in the CAM. The similarity of the tissue response of the CAM with the mammalian models, plus the low cost, simplicity, and possibility to continuously visualize the test site through the shell window make this animal model particularly attractive for the rapid in vivo screening of biomaterials.     

Leukocyte chemotaxis: A new in vivo testing technique

A new and sensitive technique is introduced for evaluating in vivo leukocyte chemotaxis. A small, disposable plastic chamber is used for holding the test solution, and an electric ink eraser for controlled abrasion of the skin test site. Another innovation is the use of capturing micropore membranes that, when placed between the test solution and the skin, collect cells for a permanent record. The small size of the chamber, the ease of application and immobilization, and the short time needed for cell collection permit experimentation with laboratory animals as well as with human volunteers. The accumulation of leukocytes in the chambers containing previously frozen or complement-activated autologous serum was 20 to 40-fold greater than in buffer controls after 3 h. Cell counts of approximately (1–2)×10³ leukocytes per mm³ were obtained in 3 h when serum diluted 11 with Hank's solution was applied to human or rabbit skin. Although predominantly polymorphonuclear leukocytes were detected in the chambers after just 3 h, prolonged incubations of 6–12 h found mononuclear cells also invading the chambers. These results clearly simulate the sequence of cellular emigration commonly observed in local inflammation.This is Scripps Clinic and Research Foundation publication number 1128. This work was supported by a United States Public Health Program Project Grant from the National Heart and Lung Institute (HL 16411) and by a Grant-in-Aid from the American Heart Association, Inc., #74-864.Dr. Hugli is the recipient of an Established Investigatorship from the American Heart Association, Inc., #72-175.     

Melanoma: A model for testing new agents in combination therapies

Treatment for both early and advanced melanoma has changed little since the introduction of interferon and IL-2 in the early 1990s. Recent data from trials testing targeted agents or immune modulators suggest the promise of new strategies to treat patients with advanced melanoma. These include a new generation of B-RAF inhibitors with greater selectivity for the mutant protein, c-Kit inhibitors, anti-angiogenesis agents, the immune modulators anti-CTLA4, anti-PD-1, and anti-CD40, and adoptive cellular therapies. The high success rate of mutant B-RAF and c-Kit inhibitors relies on the selection of patients with corresponding mutations. However, although response rates with small molecule inhibitors are high, most are not durable. Moreover, for a large subset of patients, reliable predictive biomarkers especially for immunologic modulators have not yet been identified. Progress may also depend on identifying additional molecular targets, which in turn depends upon a better understanding of the mechanisms leading to response or resistance. More challenging but equally important will be understanding how to optimize the treatment of individual patients using these active agents sequentially or in combination with each other, with other experimental treatment, or with traditional anticancer modalities such as chemotherapy, radiation, or surgery. Compared to the standard approach of developing new single agents for licensing in advanced disease, the identification and validation of patient specific and multi-modality treatments will require increased involvement by several stakeholders in designing trials aimed at identifying, even in early stages of drug development, the most effective way to use molecularly guided approaches to treat tumors as they evolve over time.     

A model for thromboembolization on biomaterials

A model was developed to describe the kinetics of protein and platelet deposition and embolization on biomaterials. The model assumes that proteins can be adequately represented by fibrinogen, albumin, and Factor XII, that protein adsorption is Langmuir-type, that surfaces are homogeneous, and that all adsorption and deposition steps are first order. Eleven model parameters were determined from literature experimental data from ex vivo experiments utilizing canine and baboon blood on Silastic, one parameter came from adsorption of Factor XII on glass, and three parameters were obtained by minimizing differences between experimental and predicted fibrinogen adsorption, and platelet deposition and embolization behavior. The model well predicted observed behavior for fibrinogen adsorption, platelet deposition, and platelet embolization on Silastic, and platelet embolization from both polyacrylamide and HEMA-MAAC.     

A new model for the artificial aorta blood vessels using double-sided radial functionally graded biomaterials

Based on radial functionally graded biomaterials and inspired by the geometry of a real aorta blood vessel, a new model was proposed to fabricate the artificial blood vessels. A finite element analyzer is employed to reach the optimal and proper material properties while earlier, it was validated by two famous theories, i.e., the first shear deformation and the plane elasticity. First, the geometry of a real ascending aorta part was simulated and then solved under the axially varying blood pressure and other real and actual conditions. Since the construction of artificial blood vessels just similar to the natural one is impossible, it was tried to find the best substitutes for other materials. Due to the significant properties of functionally graded biomaterials in the reduction in sudden changes of stress and deformation, these types of materials were selected and studied. Two types of conventional single-sided and an efficient double-sided radial functionally graded vessel were proposed and simulated. The elastic behaviors of proposed vessels were obtained and compared to ones previously attained from the real vessel. The results show that all the desired behaviors cannot be achieved by using a conventional single-sided radial FG vessel. Instead and as a conjecture, a smart double-sided radial FG biomaterial is suggested. Fortunately, the proposed material can meet all the desired goals and satisfy all of the indices simultaneously.     

Fibrin-based model for cartilage regeneration: tissue maturation from in vitro to in vivo

One of the crucial points for a successful tissue-engineering approach for cartilage repair is represented by the level of in vitro maturation of the engineered tissue before implantation. The purpose of this work was to evaluate the effect of the level of in vitro maturation of engineered cartilaginous samples on the tissue quality after in vivo implantation. Samples were obtained from isolated swine articular chondrocytes embedded in fibrin glue. The cell-fibrin composites were either cultured in vitro or directly implanted in vivo for 1, 5, and 9 weeks. Other experimental samples were precultured for either 1 or 5 weeks in vitro and then implanted in vivo for 4 additional weeks. All the samples were analyzed by histology, immunohistochemistry, biochemistry, and gene expression. The results strongly suggest that the in vivo culture in this model promoted a better tissue maturation than that obtained in the in vitro condition, and that 1 week in vitro preculture resulted in the primary structuring of the engineered composites and their subsequent maturation in vivo, without affecting the cell viability and activity, while a prolonged in vitro preculture caused a cell and matrix degeneration that could not be rescued in vivo.     

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.     

Postmortem circulation: A new model for testing endovascular devices and training clinicians in their use

The development of new medical devices, such as aortic valves, requires numerous preliminary studies on animals and training of personnel on cadavers before the devices can be used in patients. Postmortem circulation, a technique used for postmortem angiography, allows the vascular system to be reperfused in a way similar to that in living persons. This technique is used for postmortem investigations to visualize the human vascular system and to make vascular diagnoses. Specific material for reperfusing a human body was developed recently. Our aim was to investigate whether postmortem circulation that imitates in vivo conditions allows for the testing of medical materials on cadavers. We did this by delivering an aortic valve using minimally invasive methods. Postmortem circulation was established in eight corpses to recreate an environment as close as possible to in vivo conditions. Mobile fluoroscopy and a percutaneous catheterization technique were used to deliver the material to the correct place. Once the valve was implanted, the heart and primary vessels were extracted to confirm its position. Postmortem circulation proved to be essential in several of the cadavers because it helped the clinicians to deliver the material and improve their implantation techniques. Due to the intravascular circulation, sites with substantial arteriosclerotic stenosis could be bypassed, which would have been impossible without perfusion. Although originally developed for postmortem investigations, this reperfusion technique could be useful for testing new medical devices intended for living patients. Clin. Anat. 556–562, 2014. © 2013 Wiley Periodicals, Inc.     

Accelerated aging for testing polymeric biomaterials and medical devices

Elevated temperature is frequently used to accelerate the aging process in polymers that are associated with medical devices and other applications. A common approach is to assume that the rate of aging is increased by a factor of 2^ΔT/10, where ΔT is the temperature increase. This result is a mathematical expression of the empirical observation that increasing the temperature by about 10 °C roughly doubles the rate of many polymer reactions. It is equivalent to assuming that the aging process is a first order chemical reaction with an activation energy of 10R/log_e2, where R is the universal gas constant. A better approach would be to determine the activation energy for the process being considered but this is not always practicable. The simple approach does not depend on the temperature increase, provided that it is not so great that it initiates any physical or chemical process that is unlikely to be involved in normal aging. If a temperature increment θ were to increase a given polymer reaction rate n times, then an elevated temperature would increase the rate of aging by a factor of n^ΔT/θ.     

A model for the preliminary biological screening of potential keratoprosthetic biomaterials

A series of in vitro screening assays for the preliminary selection of biomaterials for use in the fabrication of artificial corneas (keratoprostheses) (KPros) have been investigated. These screening assays assessed the initial binding of inflammatory and cell adhesive proteins, activation of inflammatory proteins, adhesion of keratocytes, epithelial cells and macrophages and the production of inflammatory cytokines by keratocytes contacting biomaterials. Central optic biomaterials were selected on the basis of low-inflammatory and cell adhesion potential. Peripheral skirt materials were selected on the basis of low-inflammatory potential but good cell adhesion to anchor the implant within the host cornea. Green fluorescent protein (GFP) gene transfer was used in a novel context to investigate cell invasion in the absence of external staining techniques. Confocal laser scanning microscopy and scanning electron microscopy were used to investigate GFP positive keratocyte invasion of porous materials. The results of in vitro assays were compared to a corneal organ culture system in which the biomaterials were assessed within a stromal environment. A range of polyurethane-based interpenetrating polymers with a range of water contents were screened. All materials showed low-inflammatory potential. A reduction in biomaterial water content induced an increase in complement C3 and fibronectin binding and in cell adhesion to materials, whilst differences in co-monomer formulation had little impact. The screening methods used in the current study provide a suitable preliminary assessment regime for the in vitro evaluation of potential KPro materials.     

A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes

Acellular cartilage can provide a native extracellular matrix for cartilage engineering. However, it is difficult for cells to migrate into acellular cartilage because of its non-porous structure. The aim of this study is to establish a sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes. Cartilage from adult pig ear was cut into a circular cylinder with a diameter of approximately 6?mm and freeze-sectioned at thicknesses of 10?μm and 30?μm. The sheets were then decellularized and lyophilized. Chondrocytes isolated from newborn pig ear were expanded for 2 passages. The acellular sheets and chondrocytes were then stacked layer-by-layer, in a sandwich model, and cultured in dishes. After 4 weeks of cultivation, the constructs were then either maintained in culture for another 12 weeks or implanted subcutaneously in nude mouse. Histological analysis showed that cells were completely removed from cartilage sheets after decellularization. By re-seeding cells and stacking 20 layers of sheets together, a cylinder-shaped cell sheet was achieved. Cartilage-like tissues formed after 4 weeks of culture. Histological analyses showed the formation of cartilage with a typical lacunar structure. Cartilage formation was more efficient with 10?μm-thick sheets than with 30?μm sheets. Mature cartilage was achieved after 12 weeks of implantation, which was demonstrated by histology and confirmed by Safranin O, Toluidine blue and anti-type II collagen antibody staining. Furthermore, we achieved cartilage with a designed shape by pre-shaping the sheets prior to implantation. These results indicate that the sandwich model could be a useful model for engineering cartilage in vitro and in vivo.     

Differentiation of extracellular matrix in the cellular cartilage ("Zellknorpel") of the mouse pinna

Summary Differentiation of cellular cartilage was studied in the mouse pinna with particular reference to matrix material. Fixation of glycosaminoglycans was performed by the use of acridine orange and elastin was identified by staining thin sections with tannic acid and uranyl acetate.Condensation of mesenchymal cells (prechondroblasts) initiates the formation of a blastema of cartilaginous tissue at postnatal day 4. The synthesis of acidic glycosaminoglycans begins at postnatal day 8 when prechondroblasts transform to chondroblasts. Glycosaminoglycans can be detected within secretory vesicles of chondroblasts at postnatal day 8, in the extracellular space at postnatal day 13. Delicate collagen fibrils and elastic fiber microfibrils are seen between prechondroblasts and chondroblasts. Deposition of elastin begins at postnatal day 11. A network of elastic fibers and lamellae is formed, which replaces both collagen fibrils and elastic fiber microfibrils. In the interstice of mature cellular cartilage only elastin and proteoglycans are present (postnatal day 21).These findings indicate that cellular cartilage represents an independent kind of supporting tissue, which may serve as a progenitor of hyaline or elastic cartilage (transitional cellular cartilage) but does not differentiate from hyalin cartilage.With financial support from the Hochschuljubiläumsstiftung der Stadt Wien. Part of this work has been presented at the 3. Arbeitstagung der Anatomischen Gesellschaft, Würzburg, 6.-8. 10. 1982     

Comparison of implantation and cytotoxicity testing for initially toxic biomaterials

To evaluate the predictive value of cytotoxicity testing, the present study compares the in vivo tissue responses to in vitro cytotoxicity before and after implantation. Material toxicity was caused by addition of the toxic substance Zincdiethyldithiocarbamate (ZDEC) that is used as a standard for in vitro cytotoxicity testing. Polyurethane discs with the addition of 0.5% or 1% ZDEC as well as nontoxic discs were inserted in the abdominal wall of rats for 1 day up to 6 weeks. After explantation the foreign body response was analyzed immunohistochemically. An in vitro reanalysis of the explanted reference materials (RMs) revealed remaining high concentrations of toxic compounds after 1-week implantation, whereas no toxicity was seen after 6 weeks implantation. This was reflected in the foreign body response where a significantly thicker capsule and more inflammatory cells were seen at 1 week for the toxic implants. Over time, with decreasing toxicity, these differences disappeared. Test samples that only were subjected to in vitro extraction with water did not elute toxic compounds to the same extent as the in vivo conditions. It is concluded that many clinically useful implant materials may be unnecessarily rejected due to the results of in vitro tests.     

组织工程技术及生物材料修复运动性关节软骨损伤

目的：总结组织工程技术及生物材料在防治运动性关节软骨损伤中的应用特点。 方法：以“关节软骨,组织工程技术,生物材料”为中文关键词,以“tissue enginneering, articular cartilage, scaffold material”为英文关键词,采用计算机检索Pubmed数据库(http://www.ncbi.nlm.nih.gov/PubMed)及维普数据库(http://www.cqvip.com/)1993-01/2010-10的相关文章,排除重复研究或Meta分析类文章。以23篇文献为主,重点对修复运动性关节软骨损伤种子细胞、支架材料、细胞因子及其性能进行讨论。 结果：计算机初检得到104篇文献,根据纳入排除标准,对组织工程软骨的种子细胞、生物支架材料以及用于组织工程中的细胞因子进行总结与分析。种子细胞是制约组织工程软骨进一步临床应用的首要因素,目前常采用的种子细胞有软骨细胞、骨髓基质干细胞和胚胎干细胞等;生物支架材料包括天然材料和人工合成可降解聚合物等;用于软骨组织工程的生长因子主要包括转化生长因子、骨形成蛋白、成纤维细胞生长因子、胰岛素样生长因子等。 结论：迄今为止,无论是工程软骨的种子细胞、支架材料、培养环境等还没有任何一种材料被认为最理想,寻求一种具有良好性能的组织工程化关节软骨是未来研究的重点。但目前很多研究仍处于实验阶段,还有一些问题有待于解决,特别是组织工程细胞支架材料植入体内后,材料的降解与细胞功能发挥是否同步,会不会产生遗传物质改变、基因表达或基因突变等问题,将其应用于临床更需要相关学者专家不断的实践和探索。     

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review

Once damaged, articular cartilage has very little capacity for spontaneous healing because of the avascular nature of the tissue. Although many repair techniques have been proposed over the past four decades, none has sucessfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. Tissue engineering approaches, such as transplantation of isolated chondrocytes, have recently demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions. As such a new approach emerges, new important questions arise. One of such questions is: what kinds of biomaterials can be used with chondrocytes to tissue-engineer articular cartilage? The success of chondrocyte transplantation and/or the quality of neocartilage formation strongly depend on the specific cell-carrier material. The present article reviews some of those biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage. A new biomaterial, a chitosan-based polysaccharide hydrogel, is also introduced and discussed in terms of the biocompatibility with chondrocytes.     

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3 months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of ?25.9% and of the femur of ?21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5 mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42 days biomechanical testing yielded completely unstable conditions in the 5 mm defect femora (bending stiffness 0 N mm^?2) and a bending stiffness of 12,500 N mm^?2 in the 3 mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3 mm defects (4.83 ± 0.37 mm²), with bridging of the initial fracture defect area, compared with the 5 mm defects (2.68 ± 0.34 mm²), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5 mm defect can be considered as a critical size defect model in which biomaterials can be tested.     

A new approach to the rationale discovery of polymeric biomaterials

This paper attempts to illustrate both the need for new approaches to biomaterials discovery as well as the significant promise inherent in the use of combinatorial and computational design strategies. The key observation of this Leading Opinion Paper is that the biomaterials community has been slow to embrace advanced biomaterials discovery tools such as combinatorial methods, high-throughput experimentation, and computational modeling in spite of the significant promise shown by these discovery tools in materials science, medicinal chemistry and the pharmaceutical industry. It seems that the complexity of living cells and their interactions with biomaterials has been a conceptual as well as a practical barrier to the use of advanced discovery tools in biomaterials science. However, with the continued increase in computer power, the goal of predicting the biological response of cells in contact with biomaterials surfaces is within reach. Once combinatorial synthesis, high-throughput experimentation, and computational modeling are integrated into the biomaterials discovery process, a significant acceleration is possible in the pace of development of improved medical implants, tissue regeneration scaffolds, and gene/drug delivery systems.