Bioreactors For Bone Tissue Engineering Research Articles

Mesenchymal stem cell culture in aligned porous hydroxyapatite scaffolds using a multiwell plate bioreactor for bone tissue engineering

Regeneration of bone lost by trauma, diseases and aging, and restoration of its load-bearing function are major clinical challenges. Hydroxyapatite (HA) is a clinically proven scaffold material for bone grafting, but the random-pore structure limits the homing of the cells inside the graft and the bone regeneration progresses with the resorption of the graft material. This work is based on the hypothesis that aligned through pores in the graft will lead to a faster healing by homing the local cells inside and provide a better environment for new bone formation through the graft structure. The investigation was done using aligned porous HA scaffolds seeded with human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) and cultured in a multiwell format bioreactor setup. The cell adhesion was studied by microscopy, cell proliferation was evaluated by Alamar blue assay and osteogenic differentiation was confirmed by biochemical and molecular assays. The results indicate that the hWJ-MSCs infiltrated through the aligned porous network of the scaffold, proliferated well when cultured in the expansion medium, and differentiated into osteogenic lineage when cultured in the differentiation medium.

Design, fabrication, and characterization of a multimodal reconfigurable bioreactor for bone tissue engineering.

In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at integrating those bio‐physical stimulations that cells experience in vivo, to promote osteogenic differentiation. Nevertheless, the highly challenging combination and deployment of many stimulation systems into a single bioreactor led to the generation of several unimodal bioreactors, investigating one or at mostly two of the required biophysical stimuli. These systems miss the physiological mimicry of bone cells environment, and often produced contrasting results, thus making the knowledge of bone mechanotransduction fragmented and often inconsistent. To overcome this issue, in this study we developed a perfusion and electroactive‐vibrational reconfigurable stimulation bioreactor to investigate the differentiation of SaOS‐2 bone‐derived cells, hosting a piezoelectric nanocomposite membrane as cell culture substrate. This multimodal perfusion bioreactor is designed based on a numerical (finite element) model aimed at assessing the possibility to induce membrane nano‐scaled vibrations (with ~12 nm amplitude at a frequency of 939 kHz) during perfusion (featuring 1.46 dyn cm−2 wall shear stress), large enough for inducing a physiologically‐relevant electric output (in the order of 10 mV on average) on the membrane surface. This study explored the effects of different stimuli individually, enabling to switch on one stimulation at a time, and then to combine them to induce a faster bone matrix deposition rate. Biological results demonstrate that the multimodal configuration is the most effective in inducing SaOS‐2 cell differentiation, leading to 20‐fold higher collagen deposition compared to static cultures, and to 1.6‐ and 1.2‐fold higher deposition than the perfused‐ or vibrated‐only cultures. These promising results can provide tissue engineering scientists with a comprehensive and biomimetic stimulation platform for a better understanding of mechanotransduction phenomena beyond cells differentiation.

Optimization and Validation of a Custom-Designed Perfusion Bioreactor for Bone Tissue Engineering: Flow Assessment and Optimal Culture Environmental Conditions.

Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration of each study, the experimental setups of perfusion bioreactor systems are not always compatible with other systems. To reconcile results from different systems, the thorough optimization and validation of experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in three steps. First, an in silico modeling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterized with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimized, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported while osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress up to 15 mPa was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasizes the necessity of optimization of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.

Research progress of in vivo bioreactor for bone tissue engineering

To review the research progress of in vivo bioreactor (IVB) for bone tissue engineering in order to provide reference for its future research direction. The literature related to IVB used in bone tissue engineering in recent years was reviewed, and the principles of IVB construction, tissue types, sites, and methods of IVB construction, as well as the advantages of IVB used in bone tissue engineering were summarized. IVB takes advantage of the body's ability to regenerate itself, using the body as a bioreactor to regenerate new tissues or organs at injured sites or at ectopic sites that can support the regeneration of new tissues. IVB can be constructed by tissue flap (subcutaneous pocket, muscle flap/pocket, fascia flap, periosteum flap, omentum flap/abdominal cavity) and axial vascular pedicle (axial vascular bundle, arteriovenous loop) alone or jointly. IVB is used to prefabricate vascularized tissue engineered bone that matched the shape and size of the defect. The prefabricated vascularized tissue engineered bone can be used as bone graft, pedicled bone flap, or free bone flap to repair bone defect. IVB solves the problem of insufficient vascularization in traditional bone tissue engineering to a certain extent. IVB is a promising method for vascularized tissue engineered bone prefabrication and subsequent bone defect reconstruction, with unique advantages in the repair of large complex bone defects. However, the complexity of IVB construction and surgical complications hinder the clinical application of IVB. Researchers should aim to develop a simple, safe, and efficient IVB.

Effects of Scaffold Pore Morphologies on Glucose Transport Limitations in Hollow Fibre Membrane Bioreactor for Bone Tissue Engineering: Experiments and Numerical Modelling.

In the current research, three electrospun polycaprolactone (PCL) scaffolds with different pore morphology induced by changing the electrospinning parameters, spinning time and rate, have been prepared in order to provide a fundamental understanding on the effects pore morphology have on nutrient transport behaviour in hollow fibre membrane bioreactor (HFMB). After determining the porosity of the scaffolds, they were investigated for glucose diffusivity using cell culture media (CCM) and distilled water in a diffusion cell at 37 °C. The scanning electron microscope (SEM) images of the microstructure of the scaffolds were analysed further using ImageJ software to determine the porosity and glucose diffusivity. A Krogh cylinder model was used to determine the glucose transport profile with dimensionless variables within the HFMB. The paper discusses the roles of various dimensionless numbers (e.g., Péclet and Damköhler numbers) and non-dimensional groups of variables (e.g., non-dimensional fibre radius) on determining glucose concentration profiles, especially in the scaffold region. A negative linear relationship between glucose diffusivities across PCL scaffolds and the minimum glucose concentrations (i.e., concentration on the outer fibre edge on the outlet side (at z = 1 and r = 3.2) was also found. It was shown that the efficiency of glucose consumption improves with scaffolds of higher diffusivities. The results of this study are expected to help in optimizing designs of HFMB as well as carry out more accurate up scaling analyses for the bioreactor.

Computational modeling of media flow through perfusion-based bioreactors for bone tissue engineering.

Bioreactor system has been used in bone tissue engineering in order to simulate dynamic nature of bone tissue environments. Perfusion bioreactors have been reported as the most efficient types of shear-loading bioreactor. Also, combination of forces, such as rotation plus perfusion, has been reported to enhance cell growth and osteogenic differentiation. Mathematical modeling using sophisticated infrastructure processes could be helpful and streamline the development of functional grafts by estimating and defining an effective range of bioreactor settings for better augmentation of tissue engineering. This study is aimed to conduct computational modeling for newly designed bioreactors in order to alleviate the time and material consuming for evaluating bioreactor parameters and effect of fluid flow hydrodynamics (various amounts of shear stress) on osteogenesis. Also, biological assessments were performed in order to validate similar parameters under implementing the perfusion or rotating and perfusion fluid motions in bioreactors' prototype. Finite element method was used to investigate the effect of hydrodynamic of fluid flow inside the bioreactors. The equations used in the simulation to calculate the velocity values and consequently the shear stress values include Navier-Stokes and Brinkman equations. It has been shown that rotational fluid motion in rotating and perfusion bioreactor produces more velocity and shear stress compared with perfusion bioreactor. Moreover, implementing the perfusion together with rotational force in rotating and perfusion bioreactors has been shown to have more cell proliferation and higher activity of alkaline phosphatase enzyme as well as formation of extra cellular matrix sheet, as an indicator of bone-like tissue formation.

Fluid flow in an enclosed cavity of rotational bioreactor for bone tissue engineering

The developed mathematical model was applied for study of fluid dynamics in a rotational bioreactor for bone tissue engineering by in vitro technology. The research goal is finding an optimal mode for rotation ensuring proper cyclic loading from fluid upon the cell-seeded biomaterial. The basis for developing a mathematical model of a bioreactor was a design of rotational type biological reactor used in medical research; the liquid flow is generated through viscosity mechanism due to surface rotation. Mathematical description of flow in a reactor cavity was performed with Navier—Stokes equations. It was assumed that flow regime in the boundary layer is laminar. Numerical algorithm was accomplished using a fluid flow solver “Fluent” in the code package ANSYS-12. Four variants of generating the rotational motion in the reactor cavity were considered. A series of parametric computations was performed for the rotation frequency f in the range 0.05 ≤ f ≤ 0.25 Hz. The paper offers visualization of velocity fields in the vertical plane. The distributions for shear stress and pressure in the working zone of reactor were calculated and analyzed. Simulations demonstrated that a method of fluid rotation by driving the outer cylinder with an offset axis is the best for arranging a cyclic pressure and cyclic shear stress on the biological material.

Biomimetic characterization reveals enhancement of hydroxyapatite formation by fluid flow in gellan gum and bioactive glass composite scaffolds

This study presents production and biomimetic characterization of macroporous composite scaffolds based on gellan gum and nanoparticulate bioactive glass (GG-BAG) under conditions relevant for bone tissue engineering. Formation of hydroxyapatite (HAp) within the scaffolds was investigated in the simulated body fluid (SBF) during 14 days in two biomimetic bioreactors: perfusion bioreactor (1.1 ml/min SBF flowrate) and a bioreactor with coupled dynamic compression and SBF perfusion (5% strain, 0.68 Hz, 1 h on/1 h off, 1.1 ml/min SBF flowrate). HAp formation was evaluated by scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDS) analysis, Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (XRPD). The superficial SBF velocity of 100 μm/s in perfusion bioreactors induced the formation of abundant cauliflower-like HAp crystals throughout the scaffold interior and flake-like crystals on external surfaces resulting in improved mechanical properties as compared to the initial scaffolds. The obtained results indicate potentials of macroporous GG-BAG scaffolds in conjunction with perfusion bioreactors for bone tissue engineering demonstrating high bioactivity suited for cultures of osteogenic cells.

Bone tissue engineering in the greater omentum is enhanced by a periosteal transplant in a miniature pig model.

Reconstruction of bone defects with autologous grafts has certain disadvantages. The aim of this study is to introduce a new type of living bioreactor for engineering of bone flaps and to evaluate the effect of different barrier membranes. Scaffolds loaded with bone morphogenetic proteins and bone marrow aspirate wrapped with either a collagen membrane or a periosteal flap were implanted in the greater omentum of miniature pigs. Both histological and radiographic evaluation showed proven bone formation and increased density after 8 and 16 weeks, with an enhanced effect of the periosteal transplant. The greater omentum is a suitable bioreactor for bone tissue engineering. Endocultivation is both an innovative and promising approach in regenerative medicine.

Responses of MSCs to 3D Scaffold Matrix Mechanical Properties under Oscillatory Perfusion Culture.

Both fluid shear stress and matrix stiffness are implicated in bone metabolism and functional adaptation, but the synergistic action of these mechanical cues on the biological behaviors of mesenchymal stem cells (MSCs) is still not well-known. In the present work, a homemade oscillatory flow device was applied to investigate the effects of matrix stiffness on MSCs survival, distribution, and osteogenic differentiation in three-dimensional (3D) conditions. Furthermore, the flow field and cell growth in this bioreactor were theoretically simulated. The results demonstrated that oscillatory shear stress significantly increased the viability and distribution uniformity of MSCs throughout the scaffold after culture for 3 weeks. Compared to static culture, oscillatory shear stress could promote the collagen secretion, mineral deposits, and osteogenic differentiation of MSCs. The findings obtained from this work indicate that the oscillatory perfusion not only provides a higher survival rate and a more uniform distribution of cells but also facilitates osteogenic differentiation of MSCs. Oscillating perfusion bioreactor culture of MSCs in 3D scaffold with optimal matrix stiffness could offer an easy-to-use but efficient bioreactor for bone tissue engineering.

Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor

The use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein-2 (BMP-2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well-established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805-1813. © 2016 Wiley Periodicals, Inc.

The optimal combination of substrate chemistry with physiological fluid shear stress

Osteoblasts on implanted biomaterials sense both substrate chemistry and mechanical stimulus. The effects of substrate chemistry alone and mechanical stimulus alone on osteoblasts have been widely studied. This study investigates the optimal combination of substrate chemistry and 12dyn/cm2 physiological flow shear stress (FSS) by examining their influences on primary rat osteoblasts (ROBs), including the releases of ATP, nitric oxide (NO), and prostaglandin E2 (PGE2). Self-assembled monolayers (SAMs) on glass slides with –OH, –CH3, and –NH2 were employed to provide various substrate chemistries, whereas a parallel-plate fluid flow system produced the physiological FSS. Substrate chemistry alone exerted no observable effects on the releases of ATP, NO, and PGE2. Nevertheless, when ROBs were exposed to both substrate chemistry and FSS, the ATP releases of NH2 were upregulated about 12-fold compared to substrate chemistry alone, while the ATP releases of CH3 and OH was similarly increased 7-fold at the peak. Similar trends were observed for the releases of NO and PGE2. The expressions of ATP, NO, and PGE2 followed the pattern of NH2-FSS>Glass-FSS>CH3-FSS≈OH-FSS. ROBs on NH2 produced the optimal combination of substrate chemistry with the physiological FSS. The F-actin organization and focal adhesion (FA) formation of ROBs on various SAMs without FSS were examined. NH2 produced the best results whereas CH3 and OH produced the worst ones. Inhibition of FAs and/or disruption of F-actin significantly decreased the releases of FSS-induced PGE2, NO, and/or ATP. Consequently, a mechanism was proposed that the best F-actin organization and FA formation of ROBs on NH2 lead to the optimal combination of substrate chemistry with the 12dyn/cm2 physiological FSS. This mechanism gives guidance for the design of implanted biomaterials and bioreactors for bone tissue engineering.

Flow dynamics characterisation of a novel perfusion-type bioreactor for bone tissue engineering

Flow dynamics characterisation of a novel perfusion-type bioreactor for bone tissue engineering

Mesenchymal stem cell culture in convection-enhanced hollow fibre membrane bioreactors for bone tissue engineering

Preparation of tissue engineered bone constructs to repair large size defects is limited by the difficult supply of oxygen and nutrients to cells deep inside the constructs. Hollow fibre membrane bioreactors (HFMBs) with medium flowing in membrane lumen have structural and functional analogy with cortical bone. In fact, membranes resemble the Haversian canals and provide for a distributed and delocalized source of nutrients and oxygen to surrounding cells. HFMBs where oxygen and nutrients diffuse to cells have been proposed for bone tissue engineering. HFMBs may also be so operated as to promote spontaneous Starling flows to enhance nutrients transfer to cells. In this study, the effect of low and high Starling flows was investigated on sheep mesenchymal stem cells (shMSC) cultured in the extracapillary space of HFMBs with respect to cell distribution, proliferation and early differentiation. After 12 days of culture, at low Starling flows cells formed thin layers around the membranes uniformly along the bioreactor. At high Starling flows, cells proliferated well and formed thick multilayer aggregates filling the space among membranes at the bioreactor outlet. These results make it possible to postulate the use of HFMBs for obtaining tissue engineered constructs for the repair of large bone defects.

Bioreactors for Bone Tissue Engineering

Bone tissue engineering is a promising solution for patients with bone defects that require reconstruction. This regenerative therapy consists in culturing osteogenic cells on a biodegradable substrate to obtain a bio-hybrid construct that will stimulate bone healing after implantation. This multidisciplinary technology nevertheless requires further development before it can become routine clinical practice. One challenge is to achieve three-dimensional seeding and osteogenic commitment of mesenchymal stem cells on biomaterials under sterile and reproducible conditions. For this purpose, different dynamic culture systems have been developed. This paper reviews recent advances in the field of bioreactors for bone tissue engineering. The purpose of such systems is to improve nutrient delivery to the cells and generate shear stress that may promote cell differentiation into osteoblastic phenotypes. A brief overview of the value of computational fluid dynamics for understanding the cell environment is also provided. Finally, some proposals are made regarding the use of bioreactors as safe and controllable devices that will help commit cells and biomaterials for the regeneration of bone tissue.

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.

Perfusion Enhances Solute Transfer into the Shell of Hollow Fiber Membrane Bioreactors for Bone Tissue Engineering

Preparation of tissue engineered (TE) 3D constructs to repair large bone defects is limited by the difficult supply of nutrients and oxygen to cells in the innermost regions of constructs cultured in bioreactors. Poor oxygenation negatively affects cell viability and function. Bioreactor design optimization may help relieve these limitations. Bioreactors in which cells are cultured outside bundles of hollow fiber membranes (HFMBs) are structurally similar to natural bone. HFMB operation in pure diffusion has been reported to suffice for fibroblasts, but is deemed insufficient for bone cells. In this paper, the effect of perfusion flows in the cell compartment on solute transfer was investigated in HFMBs differing in design and operating conditions. HFMBs were designed and operated using values of non-dimensional groups that ensured solutes transfer towards the cell compartment mainly by diffusion; in the presence of low to high Starling flows; in the presence of pulsatile radial flows obtained by periodically stopping the solution flow leaving the bioreactor using a pinch valve. Distribution of matter in cell-free HFMBs was evaluated with tracer experiments in an optimized apparatus. Effectiveness of solute transfer to cell compartment was assessed based on the bioreactor response in terms of the shell volume actively involved in mass transfer (V(MTA)) according to transport models developed specifically for the purpose. V(MTA) increased with increasing Starling flows. In the pulsatile radial flow mode, tracer concentration in the shell increased 3 times faster than at high Starling flows. This suggests that controlled perfusion flows in HFMBs might enable the engineering of large TE bone constructs.

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three-dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast-like MC 3T3-E1 cells were 3D-cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell-seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering.

Oscillatory perfusion seeding and culturing of osteoblast‐like cells on porous beta‐tricalcium phosphate scaffolds

Perfusion culture systems have proven to be effective bioreactors for constructing tissue engineered bone in vitro, but existing circuit-based perfusion systems are complicated and costly for conditioned culture due to the large medium volume required. A compact perfusion system for artificial bone fabrication using oscillatory flow is described here. Mouse osteoblast-like MC 3T3-E1 cells were seeded at 1.5 x 10(6) cells/100 microL and cultured for 6 days in porous ceramic beta-tricalcium phosphate scaffolds (10 mm in diameter, 8 mm in height) by only 1.5 mL culture media per scaffold. The seeding efficiency, cell proliferation, distribution and viability, and promotion of early osteogenesis by both a static and an oscillatory perfusion method were evaluated. The oscillatory perfusion method generated higher seeding efficiency, alkaline phosphatase activity, and scaffold cellularity (by DNA content) after 6 days of culture. Stereomicroscopic observation of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining and Calcein-AM/propidium iodide double staining also demonstrated homogeneous seeding, proliferation, and viability of cells throughout the scaffolds in the oscillatory perfusion system. By contrast, the static culture yielded polarized seeding and proliferation favoring the outer and upper scaffold surfaces, with only dead cells in the center of the scaffolds. Thus, these results suggest that the oscillatory flow condition not only allow a better seeding efficiency and homogeneity, but also facilitates uniform culture and early osteogenic differentiation. The oscillatory perfusion system could be a simple and effective bioreactor for bone tissue engineering.

10204 OSCILLATORY PERFUSION SEEDING AND CULTURE FOR BONE TISSUE ENGINEERING

A compact perfusion system with oscillatory flow was developed for bone tissue engineering, and its seeding and culture effects were evaluated in present study. Mouse osteoblast-like cells, MC 3T3-E1, were seeded and cultured for 6 days in porous ceramic scaffolds by either static or oscillatory perfusion method. Compared with static culture, the oscillatory perfusional method showed significantly higher seeding efficiency and higher scaffold cellulary by DNA content analysis, as well as higher ALP activity and after 6 days of culture. Stereomicroscopy observation of MTT staining and Hoechst/PI double staining also demonstrated homogeneous seeding, proliferation and viability spatially in oscillatory system ; on contrast, static culture showed a polarized seeding and proliferation favoring only the outer and upper surface, with cells in central part of scaffold underwent apoptosis. The results demonstrated the novel oscillatory perfusion system could be a simple but useful bioreactor for bone tissue engineering with easier operation.