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.     

Fabrication and mechanical properties of PLLA and CPC composite scaffolds

The brittleness and insufficient strength of biomaterials such as calcium phosphate cement (CPC) limit their applications in physiologically non-load-bearing bone lesions. These limitations stimulated the research for developing degradable polymer-ceramic composite materials that can closely match the modulus of bones. In this study, poly (L-lactic acid)/calcium phosphate cement (PLLA/CPC) composite scaffolds were fabricated via a four-step process, namely, measurement, prototyping, compounding, and dissolving. The design and mechanical properties of the PLLA/CPC composite structures were theoretically and experimentally studied. The PLLA/CPC scaffold improved the mechanical properties of the CPC. The CPC??s compressive strength and strengthening percentage increase with higher PLLA volume. Such composites may have a clinical use for load-bearing bone fixation.     

快速成形中PAN基碳纤维/HA力学性能模拟研究

在人工骨支架增材制造过程中,为了制造出具有不同力学性能的人造骨支架适合不同病患,提出利用分子动力学(Molecular dynamics,MD)模拟方法,研究所用基体复合材料(聚丙烯腈(Poly acrylonitrile,PAN)基碳纤维和羟基磷灰石(Hydroxyapatite, HA))表面之间的相互作用及力学性能。分别在原子模拟凝聚相优化分子势能力场(Condensed-phase optimized molecular potentials for atomistic simulation studies,COMPASS)和通用力场(Universal force field,UFF)下,对比计算HA和PAN基碳纤维/HA的力学性能,并比较其弹性模量和泊松比,证明羟基磷灰石是一种各向异性材料,并给出羟基磷灰石中添加PAN基碳纤维可以有效改善其力学性能。针对沿不同剪切方向上的PAN基碳纤维/羟基磷灰石进行动力学计算,表明在不同的力场下,羟基磷灰石均是在沿(110)剪切面上与聚丙烯腈纤维产生的结合能值较大。结合径向分布函数分析,揭示了PAN/HA复合材料主要是通过PAN中的N原子与HA表面发生强作用,从而提高PAN/HA基体力学性能。     

Functional graded scaffold of HDPE/HA prepared by selective laser sintering: microstructure and mechanical properties

This paper presents a study on the effect of hydroxyapatite (HA) content on the microstructure and mechanical properties of high-density polyethylene (HDPE)/HA composites and the fabrication of functional graded scaffold of HDPE/HA by selective laser sintering (SLS). The microstructure of the sintered composite scaffolds had interconnected pores with diameters of 30–180 μm and porosity of 45–48 %. The HDPE/HA composite scaffolds had a flexural modulus of 36–161 MPa and ultimate strength of 4.5–33 MPa. The maximum loss modulus peak tended toward lower temperature values for HDPE/HA composites with 10 and 20 % of HA content, indicating that the α_χ relaxation was slightly affected by higher quantities of HA. The HA particles reinforced the matrix and minimized the plastic and definitive deformation under the test conditions. HDPE/HA functional graded scaffold fabricated using SLS with controlled microstructure and properties showed considerable potential for biomedical applications, being suitable for bone and cartilage tissue engineering.     

In-situ engineering of cartilage repair: a pre-clinical in-vivo exploration of a novel system

This investigation explores a new cartilage repair technique that uses a novel method to secure a non-woven multifilamentous scaffold in the defect site after microfracture. The hypothesis is that a scaffold provides a larger surface area for attachment and proliferation of the mesenchymal stem cells that migrate from the bone marrow. Two in-vivo studies were undertaken in an ovine model. The first study, which lasted for 8 weeks, aimed to compare the new technique with microfracture. Chondral defects, 7 mm in diameter, were created in both femoral medial condyles of five ewes. One defect was treated with the new technique while the contralateral knee was treated with microfracture alone. The results revealed that the quantity of repair tissue was significantly greater in the defects treated with the new system. The second study had two time points, 3 and 6 months, and used 13 ewes. In this study, both defects were treated with the new technique but one received additional subchondral drilling in order to stimulate extra tissue growth. The majority of the implants had good tissue induction, filling 50-100 per cent of the defect volume, while the compressive modulus of the repairs was in the range of 40-70 per cent of that for the surrounding cartilage. In addition, hyaline-like cartilage was seen in all the repairs which had the additional drilling of the subchondral bone.     

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。     

仿生多材料复合增强骨软骨支架的制造及性能研究

针对关节面上大面积骨软骨缺损修复过程中软骨形态恢复和力学环境恢复困难的问题,设计并制造一种新型聚乙二醇(Polyethylene glycol, PEG)/ 聚乳酸(Polylactide, PLA)/ β-磷酸三钙(β-Tricalcium phosphate, β-TCP)仿生多材料复合增强骨软骨支架。基于CT扫描数据重建的羊膝关节模型上进行仿生多材料骨软骨支架的结构设计,包括多孔定制结构和固定桩及仿生结构;以光固化成形技术与真空灌注工艺相结合制造了的多材料复合增强骨软骨支架,确定灌注温度220℃,真空度–0.08～–0.10 Pa。形貌观测表明真空灌注法能使PLA完全充满整个次级管道,力学试验发现复合材料支架的压缩强度(21.25 MPa ± 1.15 MPa)是单管道多孔生物陶瓷支架(9.76 MPa± 0.64 MPa)的2.17倍, PLA固定桩的剪切强度(16.24 MPa±1.85 MPa)是陶瓷固定桩(0.87 MPa±0.14 MPa)的18.7倍。因此,复合PLA的骨软骨支架具有显著的力学增强和固定能力,有望为大面积骨软骨缺损的修复提供新的治疗手段。     

Design of multi-scaffold fabrication system for various 3D scaffolds

Three-dimensional (3D) porous scaffolds have been fabricated recently for tissue engineering applications through solid free-form fabrication (SFF) technologies. A multi-scaffold fabrication system for the fabrication of scaffolds, such as polymer, polymer/ceramic, ceramic, and nanofiber, was designed in this study. The various components, including a dispenser with a maximum pressure of 750 kPa, a thermostat with a maximum temperature of 250°C, a high-voltage power supply with a maximum output of 60 kV, and a syringe pump with small flow control, play important roles in determining the process characteristics of scaffolds. The system can process applicable biomaterials with extremely high accuracy with a precision nozzle. Several 3D scaffolds, including PCL, PCL/PLGA/β-TCP, β-TCP, and PCL nanofibers, were fabricated. The morphology and pore size of fabricated scaffolds were observed through scanning electron microscopy. Results show that the scaffolds manufactured in this study can be effectively utilized as bone regeneration scaffolds.     

复合型人工半膝关节制造方法研究

结合反求技术、快速成形和快速模具技术,提出了一种新型钛合金/多孔陶瓷复合半膝关节系统的定制化制造方法。基于医学影像技术和反求技术进行半膝关节假体的个性化设计,通过快速成形技术制造人工关节的树脂原型,运用离心铸造和粉末烧结技术成形钛合金关节假体和多孔生物陶瓷活性人工骨。结果显示,半膝关节的重构误差小于0·25mm,能满足医学对假体精度的要求(0·5mm)。将钛合金/多孔陶瓷复合人工关节假体植入动物(狗)体内,与周围组织匹配良好,既具有足够的机械强度,又保证了假体的生物活性。因此,该复合型人工关节假体可用于修复体内大段骨缺损,通过机械重建和生物重建相结合,达到受损关节的功能重建,具有广泛的医学应用前景。     

仿生骨支架微观孔结构的构建与评价

在分析人体骨微观孔结构和影响支架性能因素的基础上,提出仿生骨支架微观孔结构的构建与评价方法。基于多约束背包问题模型的结构,以椭球体作为构建微观孔结构负模型的单元体,利用混合遗传算法求解微观孔结构的负模型;并将孔隙率和连通性作为约束条件,以保证仿生骨支架具有生物活性。通过不含微观孔的支架模型与负模型之间的布尔运算,构建含有微观孔结构的仿生骨支架模型。以支架的孔隙率、孔间的连通性、孔分布的均匀性、孔道的扭曲度和支架的比表面积作为评价指标,建立仿生骨支架微观孔结构的评价体系。基于支架微观孔结构的负模型,提出支架的孔隙率、连通性、均匀性、扭曲度和比表面积的计算方法,以实现对仿生骨支架微观孔结构的评价与优化。通过上述方法构建的仿生骨支架具有良好的生物活性、较好的力学性能以及均衡的降解速度。     

金属基/陶瓷复合双涂层的正接触应力分析

以球形压头为模型，采用I—deasCAD／CAE软件对Hertz弹性接触状态下金属基／陶瓷双涂层系统的应力分布情况进行了理论建模，并基于模型计算了在不同的涂层厚度／接触半宽度比和外涂层／过渡层／基体弹性模量比情况下的应力分布情况。双层系统具有相同的厚度和不同的弹性模量。论述了无涂层弹性半空间体的有限元分析结果与经典的Hertz接触力学解析解结果的一致性，计算结果有助于工程中陶瓷涂层的设计与应用。     

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.     

Development of composite porous scaffolds based on poly(lactide-co-glycolide)/nano-hydroxyapatite via selective laser sintering

Poly(lactide-co-glycolide) (PLGA)/nano-hydroxyapatite (nano-HAP) composite porous scaffolds with well-controlled pore architectures as well as high exposure of the bioactive ceramics to the scaffold surface were fabricated via selective laser sintering. Neat PLGA and the composite of PLGA/nano-HAP were used to obtain suitable process parameters. The effects of nano-HAP content on the microstructure and mechanical properties were investigated. The testing results showed that the compressive strength and modulus of the scaffolds were highly enhanced when the nano-HAP content reached from 0 to 20 wt%, while the mechanical properties experienced a sharp dropped with the nano-HAP content further increased. This might be due to the large reduction in polymer which decreased the interface bond strength between particles. It suggests that the introduction of nano-HAP as a reinforcing phase can improve the mechanical properties of the polymer porous scaffolds. The novel developed scaffolds may serve as a three-dimensional bone substrate in tissue engineering.     

Chondrogenic differentiation of human adipose mesenchimal stem cells: Influence of a biomimetic gelatin genipin crosslinked porous scaffold

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928–934, 2014. © 2014 Wiley Periodicals, Inc.     

Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold

Regularized phase tomography was used to image non‐calcified fibrous matrix in in vitro cell‐cultivated porous bone scaffold samples. 3D micro‐architecture of bone and bone scaffold has previously been studied by micro‐computed tomography, synchrotron radiation (SR) micro‐computed tomography and microdiffraction. However, neither of these techniques can resolve the low‐calcified immature pre‐bone fibrous structures. Skelite porous scaffold discs were seeded with osteoblasts, a combination of osteoblast and pre‐osteoclasts and, as controls, with pre‐osteoclasts only, and then cultivated for 8 weeks. They were subsequently imaged using SR propagation‐based phase contrast imaging. Reconstructions using a regularized holographic phase tomography approach were compared to standard (absorption) SR micro‐computed tomography, which show that quantitative analysis, such as volume and thickness measurements, of both the calcified fraction and the immature bone matrix in the reconstructed volumes is enabled. Indications of the effect of this type of culture on Skelite, such as change in mineralization and deposit of mature bone on the walls of the scaffold, are found. The results are verified with a histological study.     

Finite element biphasic indentation of cartilage: a comparison of experimental indenter and physiological contact geometries.

In experimental cartilage indentation studies, the indenter is typically a plane-ended or hemispherically ended cylinder and can be either porous or non-porous. Joints such as the hip and knee, however, have much higher radii of curvature than those used in experimental indentation testing. In order to interpret the results from such testing in a physiological context it is therefore useful to explore the effect of contact geometry on the pore pressure and strain distribution generated in the cartilage layer. Articular cartilage can be described as a saturated porous medium, and can be considered a biphasic material consisting of a porous, permeable solid matrix, and an interstitial fluid phase. This behaviour has been predicted in this study using the ABAQUS soils consolidation procedure. Four contact geometries were modelled: two typical experimental configurations (5 mm radii cylindrical indenter and hemispherical indenters) and two effective radii representative of the hip and knee (20 and 100 mm). A 10 per cent deformation, or a load of 0.9 kN, was applied over a ramp time of 2 s, which was then maintained for a further 100 s. The porous indenter generated less pore pressure compared with the equivalent non-porous indenter and produced higher values of compressive strain in the solid matrix. The predictions made using the cylindrical indenters, porous and non-porous, were dominated by the concentrations at the edge of the indenter and overestimated the peak compressive strain in the tissue by a factor of 21 and a factor of 14 respectively when compared with the hip model. The hemispherical indenter predicted peak strains in similar positions to those predicted using physiological radii, however, the magnitude was overestimated by a factor of 2.3 when compared with the knee and by 5.7 when compared with the hip. The pore pressure throughout the cartilage layer reduced significantly as the radius of the indenter was increased.     

Imaging of the Surface of Human and Bovine Articular Cartilage with ESEM and AFM

The surface of human and bovine articular cartilage was imaged with environmental SEM and AFM. The effective modulus of the surface, from force--distance curves obtained with AFM, remained constant at 9±2 kPa in the presence of synovial fluid. Extensive washing of the cartilage surface with phosphate buffered saline (PBS) removed a superficial gel-like layer, leaving a granular layer intact. Force--distance curves showed that the chemical and mechanical properties of the gel exposed to PBS changed over time. The effective modulus at the surface dropped from 481 to 4 kPa over an hour. The results suggest that the gel-like layer, having partly lost water through evaporation on removal from the joint, absorbs water from PBS. It becomes softer and eventually begins to dissolve. The low effective modulus of the gel-like layer in synovial fluid indicates that it is too soft to influence the surface roughness. Imprints of the surface under pressure were taken using a low viscosity dental kit. Imaging of the imprint surface indicated that the topography of the cartilage under pressure was similar to that of the surface after removal of the gel-like layer. In conclusion, imaging of articular cartilage with ESEM and AFM revealed two distinct non-fibrous layers, which are granular and gel-like, and cover the fibrous collagen matrix.     

平纹编织C/SiC复合材料的剪切性能

采用IOSIPESCU纯剪切试件,改进反对称四点弯曲装置进行循环加卸载,试验研究了CVI工艺二维平纹编织C/SiC复合材料的面内剪切特性。分析了不同应力水平下卸载模量、残余应变的变化。基于试验研究结果,将剪切应变分解为弹性应变与非弹性应变,分别分析弹性应变和非弹性应变的变化规律,给出了剪切应力应变关系表达式。试件断裂时,最窄截面位置形成平断面,断面扫描电镜分析发现,基体和界面裂纹是主要损伤机理,0°纤维束最后断裂。     

Multi-layered metal flow and formation of onion rings in friction stir welds

The friction stir welding (FSW) is achieved with an external tool consisting of a shoulder and pin. The shoulder and pin transfer the metal from the front side to the rear side in two distinct modes; the first and second modes of metal transfer, respectively. In the present study, the pin in the form of a cylindrical tool is used to analyse the metal flow during FSW in the second mode of metal transfer. Movement of the cylindrical tool transfers the metal from the front side to the rear side layer by layer. The longitudinal and transverse forces during the metal transfer are measured and the layered metal flow phenomenon has been proposed, due to stick and slip conditions. Based on the results obtained, the two modes of metal transfer as well as the formation of onion rings in friction stir welds have been explained. The present work can be used to model the process for improving tool and fixture design.     

基于形函数控制的组织工程骨架孔隙结构实体建模方法

组织工程支架在组织工程研究中起着重要作用,它不仅为特定的细胞提供结构支撑作用,而且能引导组织再生和控制组织结构。从几何建模的角度,寻找一种通用的组织工程骨架实体建模方法：采用有限元中六面体单元对实体模型进行网格剖分;利用有限元中的八结点六面体形函数将参数域中的基本孔隙单元映射为空间域中各种不规则孔隙几何单元。利用布尔并运算构建实体的造孔单元。再与整个实体轮廓模型(CT/MRI)进行逻辑差运算,就可以得到组织工程骨架模型。研究表明：通过有限元单元剖分方法,有效地解决了组织工程骨架孔隙体元的分布计算等技术问题,相对随机几何方法,具有很好的技术操作性和高效性。通过剖分单元约束和孔隙体元变形构型方法的应用,可大大提高骨组织孔隙形状的自然性。