Methods xxx (2015) xxx–xxx

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Guest Editor’s Introduction

Advanced methods for tissue engineering and regenerative medicine

The loss of an organ or its function is one of the most devastating medical conditions. While in some cases medications (such as insulin) or prosthetic devices (such as an artificial knee) can restore at least a part of the original function, in many cases the only definitive solution is organ transplant (such as the heart, lung, or liver). While providing life-saving solutions to many patients in need, organ transplants are seriously limited by the shortage of donor organs and the need for life-long immunosuppression. Tissue engineering emerged several decades ago with the goal of providing biological substitutes for our lost tissues and organs. The need for tissue repair is further increasing in our aging population, as we live longer and better than ever before. Clearly, an ideal approach to treating tissue loss due to congenital abnormality, injury or disease would be to re-establish the structure and function of the original tissue. This is exactly what tissue engineering attempts to do, by a variety of approaches. In some cases, an entire living structure is grown in vitro and implanted into the body to replace the missing or defective tissue—examples include skin, blood vessels, cartilage, bone, and ligaments. Whole organ engineering is the most recent and most ambitious development in this area. In other cases, the tissue is grown inside the body by inducing and augmenting the intrinsic regenerative processes—skin, bone, vascular supply, and heart are among tissues being repaired this way. These apparently different replacement and regeneration approaches have in fact a lot in common, as they rely on recapitulating the developmental processes using sets of tissue engineering tools: therapeutic cells (exogenous or mobilized in the body), biomaterials (supporting the formation of tissues in vitro or delivery and conditioning of the cells in vivo), and regulatory factors (provided in bioreactors and the body). In addition, approaches are being developed to build tissues using the patient’s own cells (to achieve a perfect match with the body), enhance integrative tissue repair by harnessing the inflammatory signals, and tailor the tissue repair to the patient and the defect being treated. While engineering of biological substitutes of human tissues is increasingly plausible, much more needs to be done until these definitive treatment modalities become a routine clinical practice. Implementation of advanced tissue engineering methods in regenerative medicine is critical for addressing the remaining challenges (e.g., blood perfusion of clinically sized tissues, robust derivation of human cells with regenerative ability, better control of the interactions with the host environment) and driving the further progress. This special issue of Methods is bringing to the reader some of the most interesting new methods from fourteen laboratories that are at the forefront of tissue engineering and regenerative medicine. 1046-2023/Ó 2015 Published by Elsevier Inc.

The first article deals with the key component of tissue engineering – the cells themselves. Human mesenchymal stem cells (MSC) are discussed, because of their regenerative, immunosuppressive and trophic properties. Park and colleagues [1] describe methods for increasing the efficacy of these cells, by genetic engineering, therapeutic agent incorporation, and cell surface modifications, MSCs can be modified to express therapeutic proteins, primed with drugs and nanoparticles, or functionalized with targeting moieties to regulate their homing. Another key component of basic and translational research in tissue engineering are animal models. To illustrate some recent developments, we selected an article by Tatara and colleagues that describe the modeling of infection, one of common complications associated with tissue implants [2]. The modeling of infection is analyzed from multiple aspects: animal model (small and large; selection of the infection microorganism), regenerative application (defect size and anatomical location; metabolic and immunological conditions), infectious agent (species; strain; inoculum size; vehicle for delivery), and readouts (clinical; colony counts; imaging). The next five articles describe the design, fabrication and utilization of advanced biomaterials for tissue engineering. The first article is by Keane and colleagues, who have pioneered the derivation of scaffolds from native extracellular matrix (ECM) [3]. Today, these scaffolds are fabricated from a number of different tissues, and are widely used both in tissue engineering research and in clinical applications. The preparation of ECM scaffolds involves the removal of all cellular material with the preservation of the structure, composition and mechanical properties of the matrix. Goldshmid and colleagues describe a method for the preparation of Pluronic–fibrinogen hydrogel microcapsules for bioprocessing of human MSCs in numbers sufficient for use in regenerative medicine [4]. The method involves cultivation of cell-laden microcapsules in suspension bioreactors, and harvesting the cells by reducing the temperature of the microcapsules to disassemble the polymer network. Importantly, the cell viability and cell yield were better than in alternate photo- cross-linked microcapsules. This simple and effective methodology can offer advantages for reproducible preparation of large numbers of cells. A new procedure for functionalizing collagen biomaterials for tissue engineering is described by Xiao and colleagues, for both hydrogels and porous scaffolds [5]. The article provides detailed protocols for modification of collagen scaffolds with growth factors and peptides, using covalent immobilization. The method involves quick handling of the material, which is critical for effective immobilization. To demonstrate that these scaffolds provide cell-instructive environments, the biomaterials were characterized in detail


Guest Editor’s Introduction / Methods xxx (2015) xxx–xxx

with respect to the efficiency of immobilization, release profiles, ultrastructure, mechanical strength, and cell responses. A new type of hydrogel, described by Ungerleider and colleagues [6], provides the biochemical composition and biomechanical properties suitable for treating myocardial infarction and peripheral artery disease, along with the ability to be delivered by injection. The method capitalizes on the minimally invasive delivery of the solution that has ability to self-assemble into a nanofibrous structure once injected and exposed to physiological temperature, and can enhance homing of the host cells to the site of injury. Notably, the hydrogel is derived from native extracellular matrix of the heart and skeletal muscle. The method allows removal of most of the tissue cells and preservation of most of the constituents of the native ECM. A different indication—traumatic spinal cord injury—required the development of a different hydrogel. Führmann and colleagues [7] have been studying the consequences of spinal cord injury and the possible treatment modalities to restore function at the lesion site. The method they describe attempts to provide controllable and sustained local delivery of therapeutic factors. To this end, a new hydrogel was designed to provide specific functions and to be injectable into the spinal cord. The material is based on hyaluronic acid functionalized by click-crosslinking, and was shown to release a brain derived neurotrophic factor. The second half of the articles in this special issue report methods for engineering various types of tissues of scientific and clinical interest. Shandalov and colleagues describe a step-by-step technique for fabrication of a vascularized muscle flap for use in reconstructions of full-thickness defects of the abdominal wall [8]. Porous polyester scaffolds were seeded with endothelial cells, fibroblasts and myoblasts to form a cellularized graft that was vascularized by implantation around the femoral artery and vein, and then transferred to the abdominal wall. A thick and vascularized tissue formed within just 1 week. The method results in a muscle flap can be used to reconstruct abdominal wall defects. Morin and colleagues describe a method for unbiased, automated quantification of microvascular networks by image analysis of histological sections [9]. At this time, two important parameters—the recruitment of supporting cells and the alignment of newly formed microvessels are measured manually. The article describes two programs for automated image analysis of the cross sections and whole mount preparations. The method quantifies standard parameters of the vascular networks, the support cell recruitment and microvascular network alignment, and shows excellent agreement with the standard manual measurements. Engineering of physiologically relevant adipose tissues for soft tissue regeneration requires perfusion, which is a challenge not addressed thus far. Abbott and colleagues describe two separate methods for perfusion cultures of engineered adipose tissues that allow long-term maintenance of tissue in culture [10]. The authors hypothesized that silk protein scaffolds (providing a 3D framework), and the perfusion flow (providing mass transport and physical signaling), support the engineering of adipose tissue, and provide data to confirm this hypothesis. In the article by Corvelli and colleagues, hyaluronic acid (HA), a component of the joint environment that is depleted in osteoarthritis (OA), was used to provide relief of joint pain [11]. To this end, cartilage models were developed to evaluate friction properties of candidate lubricants under conditions resembling mechanically induced OA. The lubricating effects of HA on articular cartilage surfaces were compared to those of synovial fluid, to determine that HA most effectively reduced friction while mimicking the friction characteristics of the OA synovial fluid under OA conditions.

Mosher and colleagues reported methods for deriving scaffolds for engineering composites of hard and soft tissues [12]. The specific challenge addressed through scaffold design was the establishment of functional integration between the host bone and bioengineered anterior cruciate ligament (ACL), with minimal stress concentration and enhanced load transfer. The new scaffold mimics the ACL-fibrocartilage-bone regions through stratified layer-by-layer organization, and was shown to support cellular organization and matrix heterogeneity of native tissues. Roach and colleagues report a method for fabrication of tissueengineered osteochondral grafts for restoring large articulating surfaces [13]. A step-by-step process is described for fabricating osteochondral constructs that precisely recapitulate the correct anatomy and topology of the joint. The method is based on a combination of high-resolution imaging, rapid prototyping, impression molding, and injection molding, to yield bi-layered cartilage tissue constructs with accurate contours, thickness, and architecture. In the last article, Bhumiratana and Vunjak-Novakovic report the method for engineering clinically sized and mechanically functional cartilage interfacing with the bone substrate, from adult human MSCs [14]. The method employs condensation and fusion of MSCs, and it forms the cartilage layer on the bone substrate. By image-guided fabrication, the osteochondral constructs were engineered in anatomically precise shapes and sizes. For the first time, the Young’s modulus and the friction coefficient of human cartilage engineered from MSCs reached physiological levels for adult human cartilage. We expect that these articles will be of interest to the readership of Methods, and the scientists, bioengineers and clinicians working in tissue engineering and regenerative medicine. References [1] J. Park, S. Suryaprakash, Y. Lao, K. Leong, Methods (2015), 10.1016/j.ymeth.2015.03.002. [2] A. Tatara, S. Shah, C. Livingston, A. Mikos, Methods (2015), 10.1016/j.ymeth.2015.03.025. [3] T. Keane, I. Swinehart, S. Badylak, Methods (2015), j.ymeth.2015.03.005. [4] R. Goldshmid, I. Mironi-Harpaz, Y. Shachaf, D. Selikta, Methods (2015), http:// [5] Y. Xiao, L. Reis, Y. Zhao, M. Radisic, Methods (2015), j.ymeth.2015.04.025. [6] J. Ungerleider, T. Johnson, N. Rao, K. Christman, Methods (2015), http:// [7] T. Führmann, J. Obermeyer, C. Tator, M. Shoichet, Methods (2015), http:// [8] Y. Shandalov, D. Egozi, A. Freiman, D. Rosenfeld, S. Levenberg, Methods (2015), [9] K. Morin, P. Carlson, R. Tranquillo, Methods (2015), j.ymeth.2015.03.014. [10] R. Abbott, W. Raja, R. Wang, J. Stinson, D. Glettig, K. Burke, D. Kaplan, Methods (2015), [11] M. Corvelli, B. Che, C. Saeui, A. Singh, J. Elisseeff, Methods (2015), http:// [12] C. Mosher, P. Spalazzi, H. Lu, Methods (2015), j.ymeth.2015.03.029. [13] B. Roach, C. Hung, J. Cook, G. Ateshian, A. Tan, Methods (2015), http:// [14] S. Bhumiratana, G. Vunjak-Novakovic, Methods (2015), 10.1016/j.ymeth.2015.03.016.

Gordana Vunjak-Novakovic Columbia University, United States E-mail address: [email protected] Available online xxxx

Advanced methods for tissue engineering and regenerative medicine.

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