Bioreactors for bone tissue engineering

Engineering bone tissue for use in orthopaedics poses multiple challenges. Providing the appropriate growth environment that will allow complex tissues such as bone to grow is one of these challenges. There are multiple design factors that must be considered in order to generate a functional tissue in vitro for replacement surgery in the clinic. Complex bioreactors have been designed that allow different stress regimes such as compressive, shear, and rotational forces to be applied to three-dimensional (3D) engineered constructs. This review addresses these considerations and outlines the types of bioreactor that have been developed and are currently in use.     

Bioactive composites for bone tissue engineering

One of the major challenges for bone tissue engineering is the production of a suitable scaffold material. In this review the currently available composite material options are considered and the methods of production and assessing the scaffolds are also discussed. The production routes range from the use of porogens to produce the porosity through to controlled deposition methods. The testing regimes include mechanical testing of the produced materials through to in vivo testing of the scaffolds. While the ideal scaffold material has not yet been produced, progress is being made.     

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.     

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.     

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.     

Physico-chemical characterization of functional electrospun scaffolds for bone and cartilage tissue engineering

Mimicking the zonal organization of the bone-cartilage interface will aid the production of functional osteochondral grafts for regeneration of skeletal joint defects. This study investigates the potential of the electrospinning technique to build a three-dimensional construct recapitulating the zonal matrix of this interface. Poly(lactic-co-glycolic acid) (PLGA) and PLGA-collagen solutions containing different concentrations of hydroxyapatite nanoparticles (nHAp) were electrospun on a thin layer of phosphate buffer saline solution spread on the collector in order to facilitate membrane detachment and recovery. Incorporation of increasing amounts of nHAp in PLGA solutions did not affect significantly the average diameter of the fibres, which was about 700 nm. However, in the presence of collagen, fibres with diameters below 100 nm were generally observed and the number of these fibres was inversely proportional to the ratio PLGA:collagen and proportional to the content of nHAp. PLGA membranes were rather hydrophobic, although the aqueous drop contact angles progressively fell from 125 degrees to 110 degrees when the content of nHAp was increased from 0 per cent to 50 per cent (w/v). PLGA-collagen membranes were more hydrophilic with contact angles between 60 degrees and 110 degrees; the values being proportional to the ratio PLGA:collagen and the content of nHAp. Also, the addition of nHAp from 0 per cent to 50 per cent (w/v) in the absence of collagen resulted in decreasing dramatically both the Young's modulus (Ym), from 34.3 +/- 1.8 MPa to 0.10 +/- 0.06 MPa, and the ultimate tensile strain (epsilon max), from a value higher than 40 per cent to 5 per cent. However, the presence of collagen together with nHAp allowed the creation of membranes much stiffer, although more brittle, as shown for membranes made with a ratio 8:2 and 10 per cent of nHAp, for which Ym = 70.0 +/- 6.6 MPa and epsilon max = 7 per cent.     

3D/4D printed bio-piezoelectric smart scaffolds for next-generation bone tissue engineering

Piezoelectricity in native bones has been well recognized as the key factor in bone regeneration.Thus, bio-piezoelectric materials have gained substantial attention in repairing damaged bone by mimicking the tissue’s electrical microenvironment(EM). However, traditional manufacturing strategies still encounter limitations in creating personalized bio-piezoelectric scaffolds, hindering their clinical applications. Three-dimensional(3D)/four-dimensional(4D)printing technology based on the principl...     

Wnt3a-induced ST2 decellularized matrix ornamented PCL scaffold for bone tissue engineering

The limited bioactivity of scaffold materials is an important factor that restricts the development of bone tissue engineering. Wnt3a activates the classic Wnt/β-catenin signaling pathway which effects bone growth and development by the accumulation of β-catenin in the nucleus. In this study, we fabricated 3D printed PCL scaffold with Wnt3a-induced murine bone marrow-derived stromal cell line ST2 decellularized matrix (Wnt3a-ST2-dCM-PCL) and ST2 decellularized matrix (ST2-dCM-PCL) by freeze-thaw cycle and DNase decellularization treatment which efficiently decellularized >90% DNA while preserved most protein. Compared to ST2-dCM-PCL, Wnt3a-ST2-dCM-PCL significantly enhanced newly-seeded ST2 proliferation, osteogenic differentiation and upregulated osteogenic marker genes alkaline phosphatase (Alp), Runx2, type I collagen (Col 1) and osteocalcin (Ocn) mRNA expression. After 14 days of osteogenic induction, Wnt3a-ST2-dCM-PCL promoted ST2 mineralization. These results demonstrated that Wnt3a-induced ST2 decellularized matrix improve scaffold materials’ osteoinductivity and osteoconductivity.     

Fabrication of scaffolds in tissue engineering: A review

Tissue engineering (TE) is an integrated discipline that involves engineering and natural science in the development of biological materials to replace, repair, and improve the function of diseased or missing tissues. Traditional medical and surgical treatments have been reported to have side effects on patients caused by organ necrosis and tissue loss. However, engineered tissues and organs provide a new way to cure specific diseases. Scaffold fabrication is an important step in the TE process. This paper summarizes and reviews the widely used scaffold fabrication methods, including conventional methods, electrospinning, three-dimensional printing, and a combination of molding techniques. Furthermore, the differences among the properties of tissues, such as pore size and distribution, porosity, structure, and mechanical properties, are elucidated and critically reviewed. Some studies that combine two or more methods are also reviewed. Finally, this paper provides some guidance and suggestions for the future of scaffold fabrication.     

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.     

组织工程支架的快速成形制备方法

这篇文章介绍了组织工程的概念及组织工程支架的重要性.与传统的组织工程支架制备方法相比,提出了新的快速成形技术.主要阐述了3维打印技术、熔融沉积技术及选择性激光烧结技术的原理及其在构建组织工程支架的应用及特点.     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.     

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.     

A comparison of imaging methodologies for 3D tissue engineering

Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current "gold standard" for imaging three-dimensional tissue engineered constructs, histology.     

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.     

组织工程支架的低温沉积制造工艺参数研究

低温沉积制造是一种新的快速成形技术。为了将这项技术更好地应用于组织工程支架的制造,根据支架孔隙率的要求,按照一定比例将PLGA和珍珠粉混合,配制成浆料,然后利用低温沉积制造工艺制备了网格型的组织工程支架,并得出了适于加工这种材料的一组工艺参数。主要从软件设定、浆料性质、温控调节和速度匹配4个方面入手,研究了在材料配制、分层和成形机制造等一系列工艺过程中参数对成形结果的影响。实验结果表明,制造出的支架结构规则能满足孔隙率的要求。     

Modeling of porous structures for rapid prototyping of tissue engineering scaffolds

Rapid prototyping techniques are increasingly used to build porous scaffolds for the regeneration of biological tissues. This paper deals with one of the most critical tasks involved by this option, i.e., the preparation of geometric data for layered fabrication. Compared to other existing approaches, this work aims at both reducing the required effort in interactive modeling and allowing a standard use of commercial prototyping systems without resorting to special treatments. The proposed method adds a porous structure to the geometric model of tissue surface in polygon format. The structure is of the Cartesian type and consists of an interconnected network of rectilinear channels, whose dimensions can be varied according to desired porosity and pore size. The algorithmic procedures needed for the generation of the porous structure have been implemented in a demonstrative software tool. Sample models of scaffolds have been generated and used to build prototype parts by different fabrication processes and systems.     

Lubrication regime of the contact between fat and bone in bovine tissue

Fat pads are masses of encapsulated adipose tissue located throughout the human body. Whilst a number of studies describe these soft tissues anatomically little is known about their biomechanics, and surgeons may excise them arthroscopically if they hinder visual inspection of the joint or bursa. By measuring the coefficient of friction between, and performing Sommerfeld analysis of, the surfaces approximating the in vivo conjuncture, this contact has been shown to have a coefficient of friction of the order of 0.01. The system appears to be lubricated hydrodynamically, thus possibly promoting low levels of wear. It is suggested that one of the functions of fat pads associated with subtendinous bursae and synovial joints is to generate a hydrodynamic lubricating layer between the opposing surfaces.