Edited by Rui L. Reis, Alexandra P. Marques, Lorna Stimson
This joint virtual Issue of both Advanced Healthcare Materials and the Journal of Tissue Engineering and Regenerative Medicine intends to highlight the nowadays-increasing interest on the development of Smart and Functional Biomaterials.
The search for bio-matrices that mimic the tissue environment, both at architectural and biochemical levels, is the aim of the research in many tissue engineering and regenerative medicine laboratories.
Micro/Nano technologies have been confirming the potential of bottom-up approaches to distinctively design extracellular matrix analogues with tailored morphologies and (bio)functionalities aimed at directly conduct and/or deliver the demanded cues for tissues regeneration. Therefore, in this virtual issue of both journals, several technologies that have been used to engineer regenerative microenvironments within the tissue engineering and drug delivery context are reviewed. Moreover, research works that take advantage of non-conventional processing methodologies to achieve smart and/or functional biomaterials for bone, myocardium and vascular tissue engineering are presented. Considering the demand on promoting neo-tissues vascularization, research works that explore different approaches to tackle this issue, benefiting from either biomaterials functionalization or architectural tailoring, are also reported.
As the physical sciences advance, techniques and understanding that have been developed over recent decades become applicable to more and more complex systems, making the observation and manipulation of living systems much more accessible. This progress has resulted in an increase in the application of research in the fields of materials science and biotechnology to biomedical problems. At Wiley, we recognized these developments with the recent launch of the journal Advanced Healthcare Materials in January 2012. In order to highlight some of the excellent research that is being conducted into the design and development of scaffolds for biomedical engineering,the editorial offices of Advanced Healthcare Materials and the Journal of Tissue Engineering and Regenerative Medicine have joined forces to bring you this virtual issue.
The Journal of Tissue Engineering and Regenerative Medicine was first published in 2007 as a forum for the publication of research and reviews on the development of therapeutic approaches which combine stem and progenitor cells with biomaterials and scaffolds, growth factors and other bioactive agents. The journal has an international editorial board of world-leading experts in the field of regenerative medicine who are led by Editor-in-Chief, Professor Rui Reis, Director of the 3B´s Research Group (www.3bs.uminho.pt).
Prof. Rui L. Reis, Editor-in-Chief of Journal of Tissue Engineering and Regenerative Medicine and Dr. Lorna Stimson, Deputy Editor of Advanced Healthcare Materials are pleased to present this Virtual Issue on Scaffolds for Biomedical Engineering:
Table of Contents:
Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications
Hojae Bae, Hunghao Chu,Faramarz Edalat, Jae Min Cha,Shilpa Sant, Aditya Kashyap, Amir F. Ahari, Cheong Hoon Kwon, Jason W. Nichol, Sam Manoucheri, Behnam Zamanian, Yadong Wang, Ali Khademhosseini
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.1494
Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell’s environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell–cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.
Engineering the Regenerative Microenvironment with Biomaterials
Jeffrey J. Rice, Mikaël M. Martino, Laura De Laporte, Federico Tortelli, Priscilla S. Briquez, Jeffrey A. Hubbell
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200197
Modern synthetic biomaterials are being designed to integrate bioactive ligands within hydrogel scaffolds for cells to respond and assimilate within the matrix. These advanced biomaterials are only beginning to be used to simulate the complex spatio-temporal control of the natural healing microenvironment. With increasing understanding of the role of growth factors and cytokines and their interactions with components of the extracellular matrix, novel biomaterials are being developed that more closely mimic the natural healing environments of tissues, resulting in increased efficacy in applications of tissue repair and regeneration. Herein, the important aspects of the healing microenvironment, and how these features can be incorporated within innovative hydrogel scaffolds, are presented.
Development of multilayer constructs for tissue engineering
NMS Bettahalli, N Groen, H Steg, H Unadkat, J de Boer, CA van Blitterswijk, M Wessling, D Stamatialis
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.1504
The rapidly developing field of tissue engineering produces living substitutes that restore, maintain or improve the function of tissues or organs. In contrast to standard therapies, the engineered products become integrated within the patient, affording a potentially permanent and specific cure of the disease, injury or impairment. Despite the great progress in the field, development of clinically relevantly sized tissues with complex architecture remains a great challenge. This is mostly due to limitations of nutrient and oxygen delivery to the cells and limited availability of scaffolds that can mimic the complex tissue architecture. This study presents the development of a multilayer tissue construct by rolling pre-seeded electrospun sheets [(prepared from poly (l-lactic acid) (PLLA) seeded with C2C12 pre-myoblast cells)] around a porous multibore hollow fibre (HF) membrane and its testing using a bioreactor. Important elements of this study are: 1) the medium permeating through the porous walls of multibore HF acts as an additional source of nutrients and oxygen to the cells, which exerts low shear stress (controllable by trans membrane pressure); 2) application of dynamic perfusion through the HF lumen and around the 3D construct to achieve high cell proliferation and homogenous cell distribution across the layers, and 3) cell migration occurs within the multilayer construct (shown using pre-labeled C2C12 cells), illustrating the potential of using this concept for developing thick and more complex tissues.
Scaffold/Extracellular Matrix Hybrid Constructs for Bone-Tissue Engineering
Richard A. Thibault, Antonios G. Mikos, F. Kurtis Kasper
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200209
The limited natural ability of the body to fully repair large bone defects often necessitates the implantation of a replacement material to promote healing. While the current clinical strategies to address such bone defects generally carry associated limitations, bone-tissue engineering approaches seek to minimize any adverse effects and facilitate complete regeneration of the lost tissue. Of particular interest are hybrid constructs that incorporate multiple components found within the native bone matrix to enhance the osteogenicity of biocompatible materials, which might otherwise be non-osteogenic. This Progress Report will focus on such hybrid constructs that incorporate multiple components from native bone matrix for bone-tissue engineering and will highlight the synthesis and characterization of the hybrid constructs, cellular attachment and proliferation within the constructs, in vitro osteogenicity of the constructs, and the biological response to in vivo implantation of the constructs at ectopic and orthotopic sites.
Channelled scaffolds for engineering myocardium with mechanical stimulation
Ting Zhang, Leo Q. Wan, Zhuo Xiong, Anna Marsano, Robert Maidhof, Miri Park, Yongnian Yan, Gordana Vunjak-Novakovic
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.481
The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs.
Neovascularization in Biodegradable Inverse Opal Scaffolds with Uniform and Precisely Controlled Pore Sizes
Sung-Wook Choi, Yu Zhang, Matthew R. MacEwan, Younan Xia
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200106
The formation of a stable vascular network in a scaffold is one of the most challenging tasks in tissue engineering and regenerative medicine. Despite the common use of porous scaffolds in these applications, little is known about the effect of pore size on the neovascularization in these scaffolds. Herein is fabricated poly(D, L-lactide-co-glycolide) inverse opal scaffolds with uniform pore sizes of 79, 147, 224, and 312 μm in diameter and which are then used to systematically study neovascularization in vivo. Histology analyses reveal that scaffolds with small pores (<200 μm) favor the formation of vascular networks with small vessels at high densities and poor penetration depth. By contrast, scaffolds with large pores (>200 μm) favor the formation of vascular networks with large blood vessels at low densities and deep penetration depth. Based on the different patterns of vessel ingrowth as regulated by the pore size, a model is proposed to describe vascularization in a 3D porous scaffold, which can potentially serve as a guideline for future design of porous scaffolds.
Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering
Birsen Demirbag, Pinar Y. Huri, Gamze T. Kose, Arda Buyuksungur, Vasif HasirciLoraine L.Y. Chiu1, Richard D. Weisel, Ren-Ke Li, Milica Radisic
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.292
Rapid vascularization of engineered tissues in vitro and in vivo remains one of the key limitations in tissue engineering. We propose that angiogenic growth factors covalently immobilized on scaffolds for tissue engineering can be used to accomplish this goal. The main objectives of this work were: (a) to derive desirable experimental conditions for the covalent immobilization of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) on porous collagen scaffolds; and (b) to determine whether primary endothelial cells respond to these scaffolds with covalently immobilized angiogenic factors. VEGF and Ang1 were covalently immobilized onto porous collagen scaffolds, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. To improve covalent immobilization conditions: (a) different reaction buffers [phosphate-buffered saline (PBS), distilled water, or 2-(N-morpholino)ethanesulphonic acid (MES)] were used; and (b) step immobilization was compared to bulk immobilization. In step immobilization, growth factors are applied after EDC activation of the scaffold, while in bulk immobilization, reagents are simultaneously applied to the scaffold. PBS as the reaction buffer resulted in higher amounts of VEGF and Ang1 immobilized (ELISA), higher cell proliferation rates (XTT) and increased lactate metabolism compared to water and MES as the reaction buffers. Step immobilization in PBS buffer was also more effective than bulk immobilization. Immobilized growth factors resulted in higher cell proliferation and lactate metabolism compared to soluble growth factors used at comparable concentrations. Tube formation by CD31-positive cells was also observed in collagen scaffolds with immobilized VEGF or Ang1 using H5V and primary rat aortic endothelial cells but not on control scaffolds.
Electrical Stimulation of Myoblast Proliferation and Differentiation on Aligned Nanostructured Conductive Polymer Platforms
Anita F. Quigley, Joselito M. Razal, Magdalena , Rohoullah Jalili, Amy Gelmi, Anthony Penington, Raquel Ovalle-Robles, Ray H. Baughman, Graeme M. Clark, Gordon G. Wallace, Robert M. I. Kapsa
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200102
In this study, nanostructured conductive platforms synthesized from aligned multiwalled carbon nanotubes and polypyrrole are investigated as myo-regenerative scaffolds. Myotube formation follows a linear path on the platforms coinciding with extent of nanotopography. In addition, electrical stimulation enhances myo-nuclear number and differentiation. These studies demonstrate that conductive polymer platforms can be used to influence muscle cell behaviour through nanostructure and electrical stimulation.
Exploitation of a novel polysaccharide nanogel cross-linking membrane for guided bone regeneration
Takayuki Miyahara, Myat Nyan, Asako Shimoda, Yuka Yamamoto, Shinji Kuroda, Makoto Shiota, Kazunari Akiyoshi, Shohei Kasugai
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.475
Cholesterol-bearing pullulan (CHP) nanogel is a synthetic degradable biomaterial for drug delivery with high biocompatibility. Guided bone regeneration (GBR) is a bone augmentation technique in which a membrane is used to create and keep a secluded regenerative space. The purpose of the present study was to evaluate the effects of the novel CHP nanogel membrane in GBR. Thirty-six adult Wistar rats were used and bilaterally symmetrical full-thickness parietal bone defects of 5 mm diameter were created with a bone trephine burr. Each defect was covered with the collagen membrane or the CHP nanogel membrane or untreated without any membrane. The animals were sacrificed at 2, 4 and 8 weeks and analysed radiologically and histologically. Furthermore, after incubating human serum with CHP nanogel or collagen, the amount of PDGF in the serum was measured using ELISA. New bone formation in terms of bone volume was higher in the nanogel group than in the control or collagen groups at 2 and 4 weeks. At 8 weeks, both membrane groups showed higher bone volumes than the control group. Notably, the newly-formed bone in the bone defect in the nanogel group was uniform and histologically indistinguishable from the original bone, whereas in the collagen group the new bone showed an irregular structure that was completely different from the original bone. After incubating with CHP nanogel, the amount of PDGF in the serum decreased significantly. CHP nanogel GBR membrane favourably stimulated bone regeneration, in which a unique characteristic of CHP nanogel, the storage of endogenous growth factors, was likely implicated.
Hierarchical Fibrillar Scaffolds Obtained by Non-conventional Layer-By-Layer Electrostatic Self-Assembly
Sara M. Oliveira, Tiago H. Silva, Rui L. Reis, João F. Mano
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200204
A new application of layer-by-layer assembly is presented, able to create nano/micro fibrils or nanocoatings inside 3D scaffolds using non-fibrillar polyelectrolytes for tissue-engineering applications. This approach shows promise for developing advanced scaffolds with controlled nano/micro environments, and nature and architectures similar to the natural extracellular matrix, with improved biological performance.
In situ functionalization of wet-spun fibre meshes for bone tissue engineering
Isabel B. Leonor, Márcia T. Rodrigues, Manuela E. Gomes, Rui L. Reis
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.294
Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (Si—OH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet-spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet-spun fibre meshes with Si—OH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet-spinning, using a calcium silicate solution as a non-solvent and functionalized in situ with Si—OH groups. In vitro tests, using goat bone marrow stromal cells (GBMSCs), showed that SPCL–Si scaffolds sustained cell viability and proliferation. Furthermore, high ALP activity and matrix production indicated that Si—OH groups improve cellular functionality towards the osteoblastic phenotype. Using this methodology, and assembling several wet-spun fibre meshes, 3D meshes can be developed, aiming at designing osteoconductive/osteoinductive 3D structures capable of stimulating bone ingrowth in vivo.
3D Porous Chitosan-Alginate Scaffolds: A New Matrix for Studying Prostate Cancer Cell-Lymphocyte Interactions In Vitro
Stephen J. Florczyk, Gang Liu, Forrest M. Kievit, Allison M. Lewis, Jennifer D. Wu,
Adv. Healthcare Mater., DOI: 10.1002/adhm.201100054
The treatment of castration-resistant prostate cancer (CRPC) remains palliative. Immunotherapy offers a potentially effective therapy for CRPC; however, its advancement into the clinic has been slow, in part because of the lack of representative in vitro tumor models that resemble the in vivo tumor microenvironment for studying interactions of CRPC cells with immune cells and other potential therapeutics. This study evaluates the use of 3D porous chitosan–alginate (CA) scaffolds for culturing human prostate cancer (PCa) cells and studying tumor cell interaction with human peripheral blood lymphocytes (PBLs) ex vivo. CA scaffolds and Matrigel matrix samples support in vitro tumor spheroid formation over 15 d of culture, and CA scaffolds support live-cell fluorescence imaging with confocal microscopy using stably transfected PCa cells for 55 d. PCa cells grown in Matrigel matrix and CA scaffolds for 15 d are co-cultured with PBLs for 2 and 6 d in vitro and evaluated with scanning electron microscopy (SEM), immunohistochemistry (IHC), and flow cytometry. Both the Matrigel matrix and CA scaffolds support interaction of PBLs with PCa tumors, with CA scaffolds providing a more robust platform for subsequent analyses. This study demonstrates the use of 3D natural polymer scaffolds as a tissue culture model for supporting long-term analysis of interaction of prostate cancer tumor cells with immune cells, providing an in vitro platform for rapid immunotherapy development.
Fibrous biodegradable L-alanine-based scaffolds for vascular tissue engineering
Deepta Srinath, Shigang Lin, Darryl K. Knight, Amin S. Rizkalla, Kibret Mequanint
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.1562
In vascular tissue engineering, three-dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l-alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue-engineering applications. In view of this, l-alanine-derived PEAs (dissolved in chloroform) were electrospun together with 18–30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue-engineering applications.
Direct-Write Assembly of 3D Silk/Hydroxyapatite Scaffolds for Bone Co-Cultures
Lin Sun, Sara T. Parker, Daisuke Syoji, Xiuli Wang, Jennifer A. Lewis, David L. Kaplan
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200057
3D silk/HA microperiodic scaffolds for bone tissue engineering and angiogenesis are fabricated by direct-write assembly. This approach can be used to control filament and spacing size in the scaffold to allow investigation of the effect of scaffold architecture on osteogenesis and vessel-like structure formation from stem cells and endothelial cells.
Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions
Ljupcho Prodanov, Jack J. W. A. van Loon, Joost te Riet, John A. Jansen, X. Frank Walboomers
J. Tissue Eng. Regen. Med., DOI: 10.1002/term.1600
Cells in situ are surrounded with defined structural elements formed by the nanomolecular extracellular matrix (ECM), and at the same time subjected to different mechanical stimuli arising from variety of physiological processes. In this study, using a nanotextured substrate mimicking the structural elements of the ECM and simulated microgravity, we wanted to develop a multifactorial model and understand better what guides cells in determining the morphological cell response. In our set-up, bone precursor cells from rat bone marrow were isolated and cultured on nanotextured polystyrene substrate (pitch 200 nm, depth 50 nm). Simulated microgravity was applied to the cells, using a random positioning machine (RPM). The results demonstrated that cells cultured on nanotextured substrate align parallel to the grooves and re-align significantly, but not completely, when subjected to simulated microgravity. The nanotextured substrate increased cell number and alkaline phosphatase (ALP) activity, whereas simulated microgravity decreased cells number and ALP activity. When the nanotextured substrate and simulated microgravity were combined together, the negative effect of the simulated microgravity ALP and cell number was reversed. In conclusion, absence of mechanical load in simulated microgravity has a negative effect on initial osteoblastogenesis, and nanotextured surfaces can partly reverse such a process.
Bioactive Glass Foam Scaffolds are Remodelled by Osteoclasts and Support the Formation of Mineralized Matrix and Vascular Networks In Vitro
Swati Midha, Wouter van den Bergh, Taek B. Kim, Peter D. Lee, Julian R. Jones, Christopher A. Mitchell
Adv. Healthcare Mater., DOI: 10.1002/adhm.201200140
Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol–gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP+ve) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2, 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 μm and 172 μm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.