Fabrication of combinatorial polymer scaffold libraries

We have designed a novel combinatorial research platform to help accelerate tissue engineering research. Combinatorial methods combine many samples into a single specimen to enable accelerated experimentation and discovery. The platform for fabricating combinatorial polymer scaffold libraries can be used to rapidly identify scaffold formulations that maximize tissue formation. Many approaches for screening cell-biomaterial interactions utilize a two-dimensional format such as a film or surface to present test substrates to cells. However, cells in vivo exist in a three-dimensional milieu of extracellular matrix and cells in vitro behave more naturally when cultured in a three-dimensional environment than when cultured on a two-dimensional surface. Thus, we have designed a method for fabricating combinatorial biomaterial libraries where the materials are presented to cells in the form of three-dimensional, porous, salt-leached, polymer scaffolds. Many scaffold variations and compositions can be screened in a single experiment so that optimal scaffold formulations for tissue formation can be rapidly identified. In summary, we have developed a platform technology for fabricating combinatorial polymer scaffold libraries that can be used to screen cell response to materials in a three-dimensional, scaffold format.     

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.     

用于人工骨制造的喷射成形技术

近年来,应用组织工程材料的人工骨因能适时降解并诱导成形人骨,而成为最理想的骨组织人工替代方法和治疗手段。介绍采用多喷射成形制造骨组织工程材料的人工骨,指出用此法得到的人工骨的材料种类及微观组织结构均与人骨高度相似,而采用骨水泥,或含有烧结等步骤,将不具有这种相似性;此人工骨具有与人骨的功能梯度上相一致的材料结构、几何结构和生理功能;人骨生长因子（ＢＭＰ）通过特殊处理,使这达到多维复合,并且具有缓释     

Imaging tissue engineering scaffolds using multiphoton microscopy

In this study, we combined two-photon autofluorescence and second harmonic generation imaging to investigate the three-dimensional microstructure and nonlinear optical properties of tissue engineering scaffolds. We focused on five different types of scaffold materials commonly used in tissue engineering, including: open-cell polylactic acid, polyglycolic acid, collagen composite scaffold, collagraft bone graft matrix strip, and nylon. By the use of multiphoton microscopy and a motorized stage, we obtained high resolution, spectrally resolved structural information of the scaffolds over large areas or in three-dimensions. Our results show that the nonlinear optical properties of the scaffolds will enable us to spectrally and morphologically distinguish the different types of scaffold materials investigated. We envision multiphoton microscopy to be a useful technique in tissue engineering applications in understanding the interplay between cultured cells and the scaffold materials.     

天然高聚物静电纺丝技术的研究与应用

天然高聚物的生物相容性好,可以促进细胞的粘附和生长,所以可以用来构建组织工程力架。通过静电纺丝技术得到的纳米纤维与胶原纤维等细胞支架材料的尺寸相近,因此,利用生物高分子静电纺丝作为血管、骨和皮肤等生物力架具有很好的效果。论述了静电纺丝技术的基本原理,并且就各种高分子材料的特点和具体应用展开了一定的说明,高压静电纺丝技术作为目前制备纳米级纤维的主要手段,未来将会在纺织工程、生物工程、组织工程以及农业工程等领域发挥更为显著的作用。     

同轴骨组织工程支架降解特性研究

为使抗骨结核药物在病灶区长期维持一定浓度,并促进术后缺损处的骨修复,应用羟基磷灰石、聚乙烯醇、丝素蛋白作为药物载体材料,结合自制机械式挤出装置制备同轴梯度骨组织工程支架.为使骨组织工程支架顺利植入体内,且保证支架负载的药物持续缓慢地释放,现对药物载体材料骨组织工程支架降解性能进行测试.研究发现降解10周后,无丝素蛋白的...     

骨组织工程细胞载体框架的两种快速成形新工艺

采用纳米羟基磷石灰石／胶原自组装材料，同时在成形过程中加入聚左旋乳酸以改善的成形性能，开发了两种新的制备骨组织工程细腻载体框架结构的快速成形工艺：T（组织工程材料）＋F（框架成形材料）成形工艺和低温喷射成形工艺。这两种工艺实现了材料的精确堆积，能成形200－500μm的可控大孔隙和孔隙梯度结构，获得理想的形态参数。     

In vitro models for bone mechanobiology: applications in bone regeneration and tissue engineering

Healthy bone healing is a remarkable, mechanically sensitive, scar-free process that leads rapidly to repair tissue of high mechanical quality and functionality, and knowledge of this process is essential for driving advances in bone tissue engineering and regeneration. Gaining this knowledge requires the use of models to probe and understand the detailed mechanisms of healing, and the tight coupling of biology and mechanics make it essential that both of these aspects are controlled and analysed together, using a mechanobiological approach. This article reviews the literature on in vitro models used for this purpose, beginning with two-dimensional (2D) cell culture models used for applying controlled mechanical stimuli to relevant cells, and detailing the analysis techniques required for understanding both substrate strain and fluid flow stimuli in sufficient detail to relate them to biological response. The additional complexity of three-dimensional (3D) models, enabling more faithful representation of the healing situation, can require correspondingly more sophisticated tools for mechanical and biological analysis, but has recently uncovered exciting evidence for the mechanical sensitivity of angiogenesis, essential for successful healing. Studies using explanted tissue continue to be vital in informing these approaches, providing additional evidence for the relevance of effects in biological and mechanical environments close to those in the living organism. Mechanobiology is essential for the proper analysis of models for bone regeneration, and has an exciting integrative role to play not only in advancing knowledge in this area, but also in ensuring successful translation of new tissue engineering and regenerative therapies to the clinic.     

组织工程支架三维结构点单元数据建模

组织工程支架所用材料和呈现结构是影响其性能的关键因素。以包含材料成分信息的点单元堆积所建立的三维离散模型,可以同时记录支架的几何结构信息和材料成分信息,为使用快速成形方法与数字微滴喷射技术制造具有更高精确的结构梯度和材料梯度的组织工程支架提供了数字模型。材料点单元堆积造型方法主要是基于模型分解的方式,将具有材料梯度和结构梯度的三维实体分割成一系列单元空间并制定单元值。主要分以下几个步骤：单元划分,所用单元形式、确定单元划分密度的法则;单元值量化方式以及量化值、从能量的角度进行局部误差分析。在理论研究的基础上,通过Matlab语言编写了功能强大的点单元数据建模软件,并应用该软件提供人体大段骨和颅部的点单元模型,从而证明此种方法的可行性。     

Periodontal regeneration: a challenge for the tissue engineer?

Periodontitis affects around 15 per cent of human adult populations. While periodontal treatment aimed at removing the bacterial cause of the disease is generally very successful, the ability predictably to regenerate the damaged tissues remains a major unmet objective for new treatment strategies. Existing treatments include the use of space-maintaining barrier membranes (guided tissue regeneration), use of graft materials, and application of bioactive molecules to induce regeneration, but their overall effects are relatively modest and restricted in application. The periodontal ligament is rich in mesenchymal stem cells, and the understanding of the signalling molecules that may regulate their differentation has increased enormously in recent years. Applying these principles for the development of new tissue engineering strategies for periodontal regeneration will require further work to determine the efficacy of current experimental preclinical treatments, including pharmacological application of growth factors such as bone morphogenetic proteins (BMPs) or Wnts, use of autologous stem cell reimplantation strategies, and development of improved biomaterial scaffolds. This article describes the background to this problem, addresses the current status of periodontal regeneration, including the background biology, and discusses the potential for some of these experimental therapies to achieve the goal of clinically predictable periodontal regeneration.     

有限元分析在骨支架设计中的应用

生物可降解骨支架设计一直是组织工程的重点,为满足骨支架植入后的性能要求,生物力学特性是其设计过程中首要考虑的问题之一。通过对不同孔隙率骨支架的分析,提出骨支架有效弹性模量与孔隙率和材料弹性模量之间的关系式,作为骨支架设计中的参考。分析中所用骨支架的孔隙率通过组合可控微单元体进行调整,对大量的骨支架的有限元分析表明:0.5%总体压应变下有效弹性模量与孔隙率和弹性模量之间关系是线性的以及对于不同属性材料,其弹性模量越高,随着孔隙率的增大,其有效弹性模量减小速度越快。     

Cytotoxicity and fibroblast properties during in vitro test of biphasic calcium phosphate/poly-dl-lactide-co-glycolide biocomposites and different phosphate materials

Reconstruction of bone defects is one of the major therapeutic goals in various clinical fields. Bone replacement materials must satisfy a number of criteria. Biological criteria are biocompatibility, controlled biodegradability, and osteoconductive or even osteogenic potential. The material should have a three-dimensional structure with an interconnected pore system so as to permit cell growth and transport of substances. The surface must permit cell adhesion and proliferation. Composite biomaterials have enormous potential for natural bone tissue reparation, filling and augmentation. Calcium hydroxyapatite/polymer composite biomaterials belong to this group of composites and, because of their osteoconductive and biocompatible properties, can be successfully implemented within bone tissue reparations. In this study, possible differences between BCP/DLPLG, pure BCP, and Bio-Oss materials were examined in vitro. During overnight incubations, fibroblast and fibroblast-like cells (L929, MRC5) were able to adhere, spread, and remain viable on BCP, BCP/PLGA, and Bio-Oss discs, as was evidenced by using light- and LVSEM-microscopy. Inhibiting influence over the cell growth is more pronounced in the cases of BCP usage on both cell lines--41.29% for L929 and 43.08% for MRC-5 cells. MRC-5 cells are, within the given experimental conditions, less sensitive on inhibiting effects for the materials BCP/PLGA and Bio-Oss (10.13% and 10.76%, respectively) than for the L929 cell lines (23.02% and 15.44%, respectively).     

Prediction of elasticity constants in small biomaterial samples such as bone. A comparison between classical optimization techniques and identification with artificial neural networks

Measuring the elasticity constants of biological materials often sets important constraints, such as the limited size or the irregular geometry of the samples. In this paper, the identification approach as applied to the specific problem of accurately retrieving the material properties of small bone samples from a measured displacement field is discussed. The identification procedure can be formulated as an optimization problem with the goal of minimizing the difference between computed and measured displacements by searching for an appropriate set of material parameters using dedicated algorithms. Alternatively, the backcalculation of the material properties from displacement maps can be implemented using artificial neural networks. In a practical situation, however, measurement errors strongly affect the identification results, calling for robust optimization approaches in order accurately to retrieve the material properties from error-polluted sample deformation maps. Using a simple model problem, the performances of both classical and neural network driven optimization are compared. When performed before the collection of experimental data, this evaluation can be very helpful in pinpointing potential problems with the envisaged experiments such as the need for a sufficient signal-to-noise ratio, particularly important when working with small tissue samples such as specimens cut from rodent bones or single bone trabeculae.     

Design and Simulation of Flow Field for Bone Tissue Engineering Sca old Based on Triply Periodic Minimal Surface

A novel method was proposed to design the structure of a bone tissue engineering scafold based on triply periodic minimal surface.In this method,reverse engineering software was used to reconstruct the surface from point cloud data.This method overcomes the limitations of commercially available software packages that prevent them from generating models with complex surfaces used for bone tissue engineering scafolds.Additionally,the fluid feld of the scafolds was simulated through a numerical method based on fnite volume and the cell proliferation performance was evaluated via an in vitro experiment.The cell proliferation and the mass flow evaluated in a bioreactor further verifed the flow feld simulated using computational fluid dynamics.The result of this study illustrates that the pressure value drops rapidly from 0.103 Pa to 0.011 Pa in the y-axis direction and the mass flow is unevenly distributed in the outlets.The mass flow in the side outlets is observed to be approximately 24.3 times higher thanthe bottom.Importantly,although the mean value of wall shear stress is signifcantly more than 0.05 Pa,there is stil a large area with a suitable shear stress below 0.05 Pa where most cells can proliferate well.The result shows that th inlet velocity 0.0075 m/s is suitable for cell proliferation in the scafold.This study provides an insight into the design analysis,and in vitro experiment of a bone tissue engineering scafold.     

Osteoblasts in bone tissue engineering

Osteoblasts are integral to the development, growth, function, repair and maintenance of bone. The osteoblast forms organic, non-mineralized bone matrix and is involved in complex interactions with a variety of factors, mediators and cell types. Degeneration, pathology, and trauma cause disruption and destruction of the normal skeletal environment and may lead to bone loss. There is a rise in active populations involved in trauma, elderly patients with fragility fractures and an overall increase in primary, revision and reconstructive bone and joint surgery. Despite the rapid evolution of implant technologies and bone grafting techniques, there is still a great demand for novel bone replacement strategies. Bone tissue engineering is the state of the art science with the potential to regenerate bone with natural form and function. This review presents the biology of osteoblasts and their current applications in bone tissue engineering biotechnologies and role in stem cell, bioactive factor, recombinant signalling molecule and gene therapy research.     

The use of acellular matrices for the tissue engineering of cardiac valves

Tissue-engineering approaches to cardiac valve replacement have made considerable advances over recent years and it is likely that this application will realize clinical success in the near future. Research in this area has been driven by the inadequacy of the currently available cardiac valve prostheses for younger patients who require multiple reoperations as they grow and develop. Tissue engineering has the potential to provide a valve capable of the same growth, repair, and regeneration as a natural valve and could improve outcomes for patients of all ages. Owing to the function and physical environment of the cardiac valve, the development of tissue-engineered replacements is unusual in that the biomechanical properties of the construct must dominate the biological properties in order for the valve to be functional at the time of implantation. As a result of this, conventional tissue-engineering scaffolds based on biodegradable polymers or collagen may not at present be suitable in this situation because of their initial limited strength. Research into the use of acellular xenogeneic and allogeneic matrices for tissue-engineered heart valves has consequently become extremely popular since the biomechanical properties of the valve can potentially be preserved with an optimal decellularization technique that removes the cells without damaging the matrix. A number of acellular scaffolds have already been tested clinically both unseeded and preseeded with cells and these have met with variable results. This article reviews the concepts involved and the advantages and disadvantages of the different approaches to tissue engineering a living cardiac valve.     

Fabrication and evaluation of calcium phosphate cement scaffold with controlled internal channel architecture and complex shape

The ability to have precise control over internal channel architecture, porosity, and external shape is essential for tissue engineering. The feasibility of using indirect stereo-lithography (SL) to produce scaffolds from calcium phosphate cement materials for bone tissue engineering has been investigated. The internal channel architecture of the scaffolds was created by removal of the negative resin moulds made with SL. Scanning electron microscopy (SEM) showed highly open, well-interconnected channel architecture. The X-ray diffraction examination revealed that the hydroxyapatite phase formed at room temperature in the cement was basically stable up to 850 degrees C. There was no phase decomposition of hydroxyapatite, although the crystallinity and grain size were different. The ability of resulting structure to support osteoblastic cells culture was tested in vitro. Cells were evenly distributed on exterior surfaces and grew into the internal channels of scaffolds. To exploit the ability of this technique, anatomically shaped femoral supracondylar scaffolds with 300-800 microm interconnected channels were produced and characterized.     

Resin luting materials: Tissue response in dog's teeth

The aim of this study was to evaluate radiographically and histologically the pulpal and periapical response to self‐adhesive (Rely X? Unicem) and self‐etching and self‐curing (Multilink^®) resin‐based luting materials in deep cavities in dogs' teeth. Deep class V cavities (0.5‐mm–thick dentin) were prepared in 60 canine premolars and the following materials were applied on cavity floor: Groups I/V—RelyX? Unicem; Groups II/VI—Multilink^®; Groups III/VII—zinc phosphate cement (control) and; Groups IV/VIII—gutta‐percha (control). Cavities were restored with silver amalgam. Animals were euthanized after 10 days (groups I–IV) and 90 days (groups V–VIII). Tooth/bone blocks were radiographed and processed for histopathological evaluation of pulp and periapical tissue response to the materials. All materials presented similar histopathological features and radiographic findings at both periods. The pulp tissue was intact. The apical and periapical regions and periodontal ligament thickness were normal. No inflammatory cells, resorption of mineralized tissue (dentin, cementum, and alveolar bone) or bacteria were observed. The lamina dura was intact and no areas of periapical bone rarefaction or internal/external root resorption were observed radiographically. It can be concluded that Rely X? Unicem and Multilink^® caused no adverse tissue reactions and may be indicated for cementation of indirect restorations in deep dentin cavities without pulp exposure. Microsc. Res. Tech. 78:1098–1103, 2015. © 2015 Wiley Periodicals, Inc.     

Temperature-driven processing techniques for manufacturing fully interconnected porous scaffolds in bone tissue engineering

The development of structures with a predefined multiscale pore network is a major challenge in designing tissue engineering (TE) scaffolds. To address this, several strategies have been investigated to provide biocompatible, biodegradable porous materials that would be suitable for use as scaffolds, and able to guide and facilitate the cell activity involved in the generation of new tissue regeneration. This study seeks to provide an overview of different temperature-driven process technologies for developing scaffolds with tailored porosity, in which pore size distribution is strictly defined and pores are fully interconnected. Here, three-dimensional (3D) porous composite scaffolds based on poly(epsilon-caprolactone) (PCL) were fabricated by thermally induced phase separation (TIPS) and by melt co-continuous polymer blending (MCPB). The combination of these processes with a salt leaching technique enables the establishment of bimodal porosity within the polymer network. This feature may be exploited in the development of substrates with fully interconnected pores, which can be used effectively for tissue regeneration. Various combinations of the proposed techniques provide a range of procedures for the preparation of porous scaffolds with an appropriate combination of morphological and mechanical properties to reproduce the requisite features of the extracellular matrix (ECM) of hard tissues such as bone.     

Stem cells for tissue engineering of articular cartilage

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.