Biomaterials in Preclinical Approaches for Engineering Skeletal Tissues

Chapter 11

Biomaterials in Preclinical Approaches for Engineering Skeletal Tissues Márcia T. Rodrigues1,2, Pedro P. Carvalho1,2, Manuela E. Gomes1,2 and Rui L. Reis1,2 13B’s

Research Group, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Guimarães, Portugal; 2ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal

Chapter Outline I. Introduction to Skeletal Tissue Engineering (STE) 127 II. Biomaterials for Translational Regenerative Medicine 128 Progression in Scaffold Development and Fabrication Techniques128 Role of Synthetic- and Natural-Based Scaffolds in TE and Regenerative Medicine 128 3D Templates—Scaffolds and Hydrogels 130 Natural-Based Polymers, Their Blends and Composites 131 Synthetic Polymers 133

I. INTRODUCTION TO SKELETAL TISSUE ENGINEERING (STE) Musculoskeletal disorders and diseases are the leading cause of disability in the United States and account for more than one-half of all chronic conditions in people over 50 years of age in developed countries [1]. In 2004, it was estimated that over 57  million musculoskeletal injuries were treated in health care settings and accounted for 60% of injuries of all types treated that year [1]. Also, the estimated cost in 2004 of treating all musculoskeletal injuries was $127.4 billion [1]. In coming years, these figures are likely to increase as musculoskeletal diseases occur more frequently as people age, and the life expectancy is likely to increase in the future. Thus, the socio-economic and health burden of skeletal injuries justifies the development of novel tissue engineering (TE) strategies to overcome these problems and improve the life of patients worldwide. It is, therefore, understandable why skeletal tissues have become a massive object of interest in the field of TE. Skeletal tissue engineering (STE) focuses on complex and very unique tissues, such as bone, cartilage, and ­tendons that act as the internal support of the body. Translational Regenerative Medicine. http://dx.doi.org/10.1016/B978-0-12-410396-2.00011-6 Copyright © 2015 Elsevier Inc. All rights reserved.

III. Could Bioreactors be the Missing Link for Biomechanic Function?135 IV. Scale-Up and Ready to Go Systems 136 V. Future Outcomes/Challenges 136 List of Acronyms and Abbreviations 137 References137

Bone is a dynamic connective tissue with multiple important functions, such as the ability to support the body and its motion [2], storage of important mineral supplies, and housing the marrow. Bone is a natural composite of approximately 70% hydroxyapatite together with 30% collagen fibers in a strong, three-dimensional structure. Articular cartilage has a simple architecture, composed of a unique type of cell, the chondrocyte, embedded within a dense extracellular matrix (ECM). Articular chondrocytes in adults do not divide, but they help to maintain cartilage integrity through balanced anabolic and catabolic activities. Unlike bone, cartilage is an inflexible connective tissue without blood vessels. Cartilage is also defined as aneural and hypocellular tissue [3], where normal mechanisms of tissue repair are not easily restored. Tendons are strong bands or cords of tissue connecting muscles to bones which allow the anatomic alignment of the skeleton, the transmission of tensile forces, and provide connective flexibility, which, in turn, enables body locomotion and joint stability/movement [4,5]. Tendon is mainly composed of fibroblasts/tenocytes and type I collagen fibers in an avascular, hypocellular, and biomechanically powerful 127

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FIGURE 1  Diagram of a traditional tissue engineering approach ­oriented for skeletal tissue engineering strategies. The triangular base is converted into a triangular pyramid enclosing the mechanical stimulation parameter associated with the healthy and functional skeletal tissues.

structure [6]. During normal activity, tendons are subjected to muscular pulls and inertial forces. The traditional TE approach is often described as a winning combination of cells, supportive material, and growth factors (GFs). Cells are required to establish a bridge between living tissues and scaffold materials. On the other hand, scaffolds provide physical and chemical support while damaged tissue is being regenerated. Finally, bioactive GFs are often included to stimulate local and/or implanted cell responses. More recently, emphasis has been given to a fourth parameter: mechanical stimulation, often associated with loading and mechano-responsive tissues such as skeletal tissues. The mechanical conditioning induced by bioreactors or other devices intends to mimic the native tissue’s properties of formation and integration of neotissue at the injury site (Figure 1). The successful combination of these parameters is expected to lead to a functional construct or tissue-engineered substitute that ultimately results in meliorated regenerative outcomes compared to currently available reparative procedures.

II. BIOMATERIALS FOR TRANSLATIONAL REGENERATIVE MEDICINE Although cells and GFs are key participants in a successful TE approach, this chapter will focus on the relevance of biomaterials toward a potential clinical scenario.

Progression in Scaffold Development and Fabrication Techniques The selection of a scaffold material for TE purposes is of extreme importance as the scaffold design dictates, to a great extent, the response of seeded cells and ultimately

determines the functionality of the grown tissue. Scaffold design encompasses a combination of selected materials and methods, considering a set of structure-property specifications for a targeted application. The purpose of creating different methodologies relies on the custom-designed development and combination of features and properties that are likely to direct cell responses in the most appropriate manner, considering the desired application. Overall, for a scaffold to serve as the temporary ECM, a few basic requirements are necessary and are summarized in Table 1. In the last decades, a pool of multiple techniques and methodologies for scaffolding fabrication has been described in an attempt to address and explore the major functional architectural and compositional cues of native tissues. The search for improved and tissue-oriented scaffolds extended the knowledge on biomaterials potential and highlighted the interest for multimodal scaffolds. The multifunctional or multimodal properties of these scaffolds result from the combination of different features that are not typically available in a given material, increasing their potential role in regenerative medicine strategies. In Table 2 several fabrication technologies aimed to address the needs of skeletal tissues have been summarized. Fabrication technologies such as particulate-leaching have been suggested for TE for several decades now, whereas supercritical fluid and 3D printing are more recent technologies. Some technologies produce templates at a microscale level for fiber and network development, while others use nanotechnology for tailoring orthopedic scaffolds in an attempt to mimic the ECM structure and complexity at a biologic scale. Some technologies seem to be particularly more directed into a specific skeletal tissue and are available to be explored, alone or in combination, toward other skeletal tissues. Nevertheless, these technologies may be combined to engineer scaffolds with novel structures and physicalchemical features.

Role of Synthetic- and Natural-Based Scaffolds in TE and Regenerative Medicine Biomaterials play a pivotal role as scaffolds to provide 3D templates and synthetic ECM environments for tissue regeneration. These materials can be obtained from innumerous sources from synthetic to natural materials. In the particular case of synthetic polymers, these are typically more versatile in tailoring a wide range of properties and structure features, representing a reliable source of raw materials. Synthetic polymers are described to degrade by hydrolysis of its ester linkages in physiological conditions [36,37] and to avoid immunogenicity problems [37,38], which are compelling arguments for pursuing approaches for TE and regenerative medicine purposes. Nevertheless, the majority of synthetic polymers are hydrophobic, which presents a major drawback for the migration of viable cells into the scaffold core.

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TABLE 1  Scaffold Requirements Aimed at Skeletal Tissue Engineering Scaffold Properties

Requirements

Biodegradability

Degradation kinetics compatible with rate of tissue regeneration and restoration of function

Biocompatibility

Do not elicit an immunological response Interaction with the surrounding tissues Induce host tissue integration

Structural architecture

Architectural features mimicking native tissue and addressing native tissue needs Match tissue defect size and shape Promote the easy diffusion of nutrients and cellular waste products Allow cell penetration/tissue ingrowth

Mechanical strength

Ensure scaffold integrity under (constant) loading of variable magnitude Allow active range of free movement Allow smooth and efficient gliding, in the case of tendon and cartilage tissues

Biofunctionality

Stimulate local environment through (1) cellular interactions with biomaterial surface properties, (2) release of bioactive molecules (if available in the system), and/or (3) native cell recruitment for tissue regeneration. Exhibit features with potential to stimulate/induce vascularization (bone and tendon tissue) Avoid adhesion formation, in the case of tendon tissues Assist cell infiltration, proliferation, and differentiation (if required) Assist the delivery of therapeutic molecules in growth factor strategies

Others

Easy to handle, store and sterilize Easy to package and transport to the clinical theater Ability to be cryopreserved without significantly affecting scaffold properties

Adapted from Refs. [6,7].

TABLE 2  Examples of Fabrication Technologies Described in the Production of Scaffolds for Several Skeletal Tissues

Nano

Micro-scale

Fabrication Technologies

Tendon

Bone

Cartilage

Electrospinning

[8–12]

[13]

[14,15]

Phase separation

[16]

Melt-spinning/fiber bonding

[17–19]

Freeze drying

[21]

[22–24]

Particle leaching

[16]

Supercritical fluid

[26,27]

Textile Rapid prototyping/3D bioprinting

On the other hand, natural polymers have been presented as an interesting option to the currently used synthetic materials due to a higher biodegradability rate and noncytotoxicity. Natural polymer synthesis often involves enzyme-catalyzed, chain growth polymerization reactions of activated monomers, which are typically formed within cells by metabolic processes.

[9,28,29]

[20]

[25]

[29] [30–35]

Figure 2 clearly shows the relevance and increasing interest of studying polymeric materials in the development of 3D supportive systems for STE. Independently of the origin, polymers lack properties to stimulate biological functions, such as osteoconductivity and cell bioactivity [39]. Concerning these properties, ceramic materials offer advantages over polymers, but their

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FIGURE 2  Graphic representation of the number of publications per year on the type of materials applied for bone, cartilage, and tendon tissue engineering over the past decade (using the Web of Science®, September 2013 Thomson Reuters). The data represented for 2013 accounts only for publications referenced from January to September.

high modulus and brittle behavior [40] limit their processability into 3D scaffolds [41,42]. Nevertheless, these properties can be induced by the incorporation of functional groups, such as silanol groups [43], or by the incorporation of bioactive molecule/agents [11,44–46].

3D Templates—Scaffolds and Hydrogels Scaffold definition often refers to 3D templates for TE in a solid-state form and produced from several fabrication technologies, as the ones described in Table 2. Nonetheless, some materials are versatile and can be modified into hydrogels that may also be used as scaffolding materials.

Hydrogels are cross-linked hydrophilic polymers that contain large amounts of water without dissolution. These water-rich templates offer some interesting advantages over scaffolds. For instance, hydrogels can properly fill irregular-shaped tissue defects, and the incorporation of cells or bioactive agents within the hydrogel matrix is quite easily achieved. Also, hydrogels maintain a hydrated environment suitable for cell colonization and infiltration; they can be delivered in vivo in minimally invasive manners and evidence properties similar to tissues and the ECM which are controllable by chemical crosslink densities [47]. Although scaffolds are widely applied in TE of skeletal tissues, polymers under the form of hydrogels have been more frequently applied as cartilage encapsulation systems.

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Natural-Based Polymers, Their Blends and Composites In recent years, some polymers have been more extensively studied in combination with other polymers or ceramic materials, as it became obvious that blending various natural-based polymers may overcome limitations associated to single polymers, through the augmentation of specific properties and/or by means of synergistic or magnified effect of each biomaterial feature or property. The following sections will provide an overview of some of these recent studies, focusing on their translational potential evidenced by preclinical studies outcomes. Hyaluronic Acid Hyaluronic acid (HA) is a glycosaminoglycan (GAG) present in all vertebrates. HA is a major component of connective tissues where it plays an important role in lubrication, cell differentiation, and cell growth. HA has been investigated with particular interest in bone and cartilage applications, but not so much emphasis has been given to tendon tissue. HA hydrogels have been often used as delivery systems of osteoinductive and angiogenic GFs for bone TE. Some GFs, such as bone morphogenic protein-2 (BMP-2) delivered from HA hydrogels showed a clear osteoinductive effect in a calvarian rat model [48], that in combination with or co-delivery of vascular endothelial GF increased the amount of mineralized tissue formed [48]. Despite the interesting plasticity of HA studies with preclinical outcomes, it is essential to assess the safety, tolerability, and long-term efficiency of HA in the clinical scenario. Chitosan Chitosan is obtained from crustacean shells and is a material structurally similar to GAGs, being degradable by enzymes in humans. Chitosan is a versatile polymer, and often is used as a blend in the formation of a 3D scaffold structure for bonerelated approaches. In recent studies, the preclinical model of choice has been the calvarian-induced defect in rats [49]. A porous scaffold developed from the combination of chitosan and alginate stimulated bone regeneration and exhibited interesting features, including shape-ability to fit the defect site, degradability up to 16 weeks after implantation, and the possibility to be molded into larger dimensions [49]. Cartilage studies also revealed to be successful using chitosan as a base material. Hao et al. [50] focused on the use of injectable chitosan. Since injected chitosan requires several minutes to gelify, and some gel may be misplaced out of the defect, temperature-responsive chitosan hydrogels were developed with beta-sodium glycerophosphate and hydroxyethilcellulose [50] before transplantation in vivo into sheep cartilage defects. The chitosan scaffold together

with the encapsulated chondrocytes showed the best results for cartilage regeneration after 24 weeks of implantation, as the defect was completely repaired. Another interesting publication refers the application of a chitosan-alginate electrospun barrier to prevent adhesion formation, which can severely interfere with the restoration of tissue function. Despite the fact that the aim of this study was to reduce peritoneal adhesions, the outcomes could be easily translated to tendon tissues, particularly affected by adhesion formation, and consequently with limitation in restoring local tissue function [51]. The flexibility of chitosan for application in skeletal tissues is already established by several animal models and strategies. The next step is to move into clinical studies, as clinical trials employing chitosan biomaterial for regenerative medicine are still scarce. Boynuegri et al. [52] recently publish the use of a chitosan gel in periodontal intraosseous defects of 19 patients. Regardless of the modest results, radiographic data of bone fills in the presence of chitosan (alone or in combination with demineralized bone matrix/collagenous membrane) were found to be statistically significant when compared with baseline. This pilot study evidences the potential applicability of chitosan for human periodontal regeneration, and indirectly to bone defect strategies. Alginate Alginate has been used in cell encapsulation and drug delivery systems because it gels easily, has low toxicity and is readily available. A significant disadvantage is that degradation occurs through ionic exchange with surrounding media. Although several studies on alginate applicability to regenerative medicine of skeletal tissues have been published recently, these works focus on a more proof-ofconcept approach in animal models rather than describing the potential use of alginate-based strategies in the clinical practice. An example is the development of alginate microspheres as a matrix to deliver platelet-rich plasma and adipose-derived stem cells toward bone TE [53]. Using minimally invasive engineering, the loaded spheres induced both vascularization and mineralization in a heterotopic site of a nude mice model [53]. Moreover, alginate microspheres have also been in vivo assessed for co-encapsulation of bone marrow mesenchymal stem cells (MSCs) and ­anti-BMP-2 monoclonal antibody, demonstrating bone ­tissue regeneration in a mouse critical-sized calvarian defect [54]. Agarose Agarose is a natural-based polysaccharide obtained from agar. It has been used for a variety of life science applications. Similar to alginate, scientific advances on agarose systems have been scarcely reported in the last few years. Despite the slow degradation profile and the low mechanical

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properties, the soft and flexible structure that allows the control of pore dimensions provided by these gels has been shown to recreate a better 3D environment, suitable for chondrocyte maintenance [55,56], and MSC differentiation into the chondrogenic lineage [55]. Additionally, chondrocytes embedded in agarose gels are able to sense and respond to mechanical forces [56], which could reveal an efficient system for cartilage-like substitutes. Without complex in vivo models to provide the necessary data, agarose relevance on TE applications remains unclear. Carrageenan Carrageenan is a thermally responsive material, inexpensive and easy to manipulate. The gelation is quite easy, and despite being originated from red seaweeds, carrageenan display close similarities with mammalian GAGs. This polymer is influenced not only by temperature but also by pH and cation concentration, and its degradation occurs through ionic exchanges with surrounding media. The studies on κ-carrageenan potential for skeletal regeneration are quite recent, and further investigation should be considered in forthcoming years. Nevertheless, κ-carrageenan hydrogels were shown to support the viability, proliferation, and chondrogenic differentiation of human adipose-derived stem cells [57]. Also, κ-carrageenan gels demonstrated an increase in stiffness and viscoelastic properties when cell encapsulated. This increment was verified with longer times in chondrogenic medium cultures [57]. Another study reported the development of κ-carrageenan beads incorporated with platelet-derived GF to induce the angiogenic mechanism required for bone regeneration, indicating that this polymer may also have potential for bone TE [44]. The ability of κ-carrageenan hydrogel systems to structurally withstand cryopreservation together with the effectiveness in maintaining the proliferative and chondrogenic potential of encapsulated cells [58] makes these constructs potential bioengineered products for readyto-use clinical applications. Collagen Collagen is a nano-fibrous material, being the most abundant protein in the human body. Because of collagen’s relevant role in native ECM, it has been used as a raw material for the production of scaffolds for STE. However, a major concern of using collagen in the human body is its animal origin, which may be associated with disease transmission risks. In the particular case of tendon strategies, several methods have been developed to form highly oriented and densely packed collagen bundles with mechanical strength approaching that of tendons. Collagen bundles fabricated by an electrochemical method indicated that these aligned bundles were not cytotoxic, and both tenocytes and MSCs were able to populate and migrate on electrochemically

aligned collagen bundles [59]. In another study with e­lectrochemical-aligned collagen threads, these scaffolds induced tenogenic differentiation of human MSCs, even in the absence of bioinductive cues [60]. MSCs preferred aligned threads over random threads, with increased expression of scleraxis, collagen 3, and tenomodulin genes [60]. Under osteogenic supplementation, MSCs were able to proliferate and differentiate into the osteogenic lineage in a collagen-alginate fibrous hydrogel reported for cell delivery aiming at bone TE strategies [61]. MSCs were successfully delivered to a rat calvarian model [61], and the bone healing was significantly improved in the presence of these constructs cultured under osteogenic stimulation. Multiple combinations have been described in the literature to fill the gap for a successful bone or osteochondral scaffold. These include the application of collagen with hydroxyapatite or calcium phosphates. The story of a successful nano-composite multilayered biomaterial obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles for osteochondral TE has been reported by Kon et al. [62]. In previous studies, the collagen-hydroxyapatite scaffold implanted in the femur condyles of a sheep model showed that this scaffold led to the regeneration of articular tissue with an ordered histoarchitecture [62]. Afterward, the scaffold was assessed in a pilot clinical trial with 13 patients with grade III–IV chondral and osteochondral lesions of the knee. The short-term follow-up, confirmed at 6 months by magnetic resonance imaging, has demonstrated good stability of the scaffold without any other fixation device [63]. Silk Silk is a natural material that has been used for textile production and surgical sutures for centuries because of its excellent tensile mechanical properties. Silk has a low enzymatic degradation rate, and some concerns arise on potential cytotoxic effects. Considering silk’s intrinsic mechanical properties, silk is a promising biomaterial for bone tissue strategies. The potential of porous silk fibroin scaffolds with RGD sequences [64] was explored by Hofmann et al. in calvarian nonloading defects induced in a mouse model. These scaffolds, developed with different porous dimensions and seeded with human MSCs, showed evidence of good integration, vascularization, and bone ingrowth just after 8 weeks of implantation [64]. Also, Mandal et al. investigated the use of reinforced silk microfibers to produce bone-engineered scaffolds in a mice ectopic model [65]. Although some inflammatory cells were detected in the explants, dense tissue ingrowth was observed with vascularization surrounding the implant [65]. In the case of osteoporotic defects of critical-sized dimensions, silk hybrid with calcium phosphate revealed a favorable outcome in osseointegration after local implantation. The composite scaffold produced by freeze drying

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technique assisted a remodeling process of bone formation over skeletal deterioration in a rat model [66] suggesting its supportive therapeutic potential. Silk based scaffolds were also reviewed for tendon related strategies. Silk fibroin braided scaffolds can form a 3D structure where tenocytes were able to adhere and proliferate [28]. Moreover, the neotissue formed in the induced Achilles defect after implantation resembled native tendon with bundles of collagen well oriented [28], showing potential for Achilles tendon regeneration in a rabbit model.

Synthetic Polymers Polylactic Acid (PLA)/Poly l-Lactic Acid (PLLA) Polylactic acid (PLA) is a thermoplastic, aliphatic polyester, produced from non toxic renewable feedstock, naturally occurring organic acid, or made by fermentation of sugars obtained from renewable resources such as sugarcane. PLA and its copolymers have been widely used in TE for scaffold fabrication. Aligned bundles of electrospun poly(l-lactic acid) nanofibers were shown to support stem cell expansion and tenogenic differentiation [9]. Moreover, in the presence of tenogenic GFs and under cyclic tensile strain, human MSCs differentiated into the tenogenic lineage, evidenced by the significant upregulation of scleraxis gene expression [9]. Polyglycolic Acid (PGA)/Poly l-glycolic Acid (PLGA) Polyglycolic acid (PGA) is a biodegradable, thermoplastic polymer and the simplest linear, aliphatic polyester. PGA and its copolymers have been used as a material for the synthesis of absorbable sutures and as synthetic polymers for TE applications. Although a PGA scaffold was not sufficient to promote cartilage repair in a rabbit study performed by Zhao et al. [67], the combination of nonwoven PGA fiber meshes with autologous bone marrow concentrate or with cultured stem cells from bone marrow showed improved cartilage repair 8 weeks after implantation. The full thickness defect created included more newly formed cartilage tissue and hyaline cartilage-specific ECM [67], indicating the applicability of PGA in cartilage TE. Xu et al. [68] explored the possibility of in vivo reconstruction of a tendon sheath using a hen model. For this purpose, a sheet of unwoven PGA scaffolds, seeded with tendon sheath cells, was inserted at the defect site. After 3 weeks of in vivo implantation, a neo-tendon sheath that resembled native tissue was formed underneath the subcutaneous tissue [68]. In another tendon study, dermal fibroblasts and tenocytes were cultured on unwoven PGA fibers in a porcine model for flexor digital superficial tendons. Both types of cells showed a good attachment and matrix production [69]. More interestingly, in vivo results showed that engineered tendons with both types of cells

were similar to each other in their gross view, histology, and tensile strength. After 14 and 26 weeks of implantation, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded [69]. Another study combined a PLGA scaffold approach with clinical treatment: continuous passive motion (CPM) versus immobilization. The study was performed in rabbit knees. As early as 4 weeks after implantation, the defects with PLGA scaffold exhibited better collagen alignment and higher GAG content in the core of their repaired ­tissues, particularly in under CPM. At week 12, sound osteochondral repair and hyaline cartilaginous regeneration was observed in the PLGA-CPM group with the presence of type II ­collagen expression, osteocyte maturation, and trabecular bone deposition. This study demonstrated that the association of CPM treatment together with PLGA implantation has resulted in a positive effect on osteochondral regeneration in a rabbit knee joint model [70]. Osteochondral studies combining dl-poly-lactide-coglycolide with beta-tricalcium phosphate were assessed in the clinical field [71]. Ten patients were treated with this scaffold laden with chondrocytes to repair osteochondral lesions of femoral condyles. A 24-month follow-up indicated a successful regeneration of hyaline cartilage. Polycaprolactone (PCL) Polycaprolactone (PCL) is a versatile polymer and fully biodegradable, with a degradation time of the order of two years [72]. PCL also forms compatible blends and copolymers with a wide range of polymers producing materials with unique elastomer properties [37]. For all of these reasons, PCL is a Food and Drug Administration (FDA) approved material, which has been described to have cellular compatibility with several tissue-engineered tissues, and widely studied for biomedical applications, including bone[30] and cartilage [14] oriented approaches, and long-term implantable devices. PCL is already used clinically, for instance, as a drug delivery device or suture base material [37]. However, in the last couple of years, approaches have been made toward the application of PCL in more complex systems as for STE. PCL scaffolds fabricated via selective laser sintering also showed interesting mechanical properties that may have the ability to withstand early functional loading and can be fabricated into custom-design defects, such as a mandibular condyle [30]. These PCL scaffolds were able to enhance tissue in-growth and evidenced potential to be used for skeletal tissues [30]. More recently, PCL nanofibers were thought to be used in the development of a biomimetic sheath membrane for avoiding tendon adhesions. The membrane consisted of a HA-loaded poly(ε-caprolactone) fibrous membrane as the inner layer, and a PCL fibrous membrane as the outer layer was fabricated by a

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combination of sequential and microgel electrospinning technologies [12]. The application of this flexible membrane in a repaired flexor digitorum profundus tendon of a hen model revealed that the intratendinous collagen bundles exhibited better tendon healing 3 weeks after implantation. Also, peritendinous adhesions were reduced, and tendon gliding improvement was observed indicating a preliminary success of this TE strategy for the prevention of adhesion formation. Starch and Polycaprolactone (SPCL) Scaffolds Starch is a polysaccharide produced by green plants as an energy store. It is quite abundant in nature and is an almost unlimited source and low-cost associated raw material. The combination of starch and polycaprolactone (SPCL) has resulted in different forms from particles to 3D scaffolds using fabrication techniques such as melt fiber extrusion, wet-spinning, rapid prototyping, or solvent casting. SPCL scaffolds exhibit better mechanical properties (around 2 MPa) than most scaffolds produced by other biodegradable polymers [73,74]. Furthermore, in vivo studies not only indicate a good integration of the starch-based materials in the host [18], but also that these biomaterials have a weak potential to stimulate an inflammatory reaction [75]. SPCL scaffolds have been further studied using other animal models, namely in critical bone defect models. One of the works developed focused on the relevance of these scaffolds seeded with amniotic fluid stem cells (AFSCs) under different stages of differentiation so as to select the best construct aiming at bone tissue regeneration. Despite the fact that in vivo

formation of new bone was observed under all conditions in femoral critical-sized defects in nude rats, the most complete repair of the defect was observed after 16 weeks in the animals receiving the SPCL scaffolds seeded with osteogenically committed AFSC [18] (Figure 3). The presence of blood vessels was also observed in the inner sections of the scaffolds suggesting that these cells could potentially be used to induce bone regeneration and angiogenesis in nonunion bone defects [18]. In another study, SPCL scaffolds obtained by wet-­ spinning technique were used in critical-sized calvarial defects of adult male nude mice [76]. Improved new bone deposition and osseointegration was observed in SPCL loaded with human adipose derived stem cells (hASCs) engrafted calvarial defects as compared to control groups that showed little healing [76]. SPCL wet-spun fibers confirmed to be a suitable and biocompatible material for adipose-derived stem cell loading and implantation in bone regeneration strategies. Although many natural- and synthetic-based biomaterials have been widely investigated for skeletal applications, combining fabrication techniques and sometimes other biomaterials, most of them are still confined to in vitro and later to preclinical assays, often using rodent and lagomorph models. But, recent publications suggest that the osteochondral field is one of the areas with more rapid evolution, where several studies, mainly with composite scaffolds have already advanced into clinical trials [63,71]. These are followed closely by ceramics application in bony defects, especially aimed at orthodontics regeneration. In a study involving 54 patients, beta-­tricalcium phosphate enriched with recombinant platelet-derived GF-BB

FIGURE 3  Volumetric measurements of the critical-sized defect section obtained from micro-CT analysis, representing the neobone formed area in green. The defect was either left empty (blank), filled with starch and polycaprolactone (SPCL) scaffold, or filled with the SPCL plus amniotic fluid stem cells (AFSCs). When the defect was filled with SPCL seeded with AFSCs, three stages of differentiation were considered: undifferentiated (third column), AFSCs committed to the osteogenic lineage (committed) with expression of bone associated markers, and AFSCs osteogenically differentiated (fully differentiated). In fully differentiated cells, a mineralized matrix was detected in the SPCL-AFSCs constructs implanted. The two time points selected in this experiment were 4 and 16 weeks after implantation. The SPCL scaffold was produced by melt-spinning followed by a fiber bonding technique. Reproduced with permission from Ref. [18].

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was assessed in periodontal osseous defects [77]. The combination resulted in an increment in bone formation and soft tissue healing. No adverse effects were observed in any of those patients during the follow-up. Despite the limited clinical evidence that scaffolds can be used safely and effectively in regenerative approaches for skeletal tissues, the limited clinical trials reported so far have supported preclinical models, and thus have produced promising results. Table 3 summarizes the biomaterials described in this section to produce scaffolds and hydrogels for tendon, bone, and cartilage engineered strategies. Some biomaterials are more versatile and were assessed for several skeletal tissues, whereas others are more extensively explored for a specific tissue strategy. The table discloses compiled information from works published in the last 5 years.

III. COULD BIOREACTORS BE THE MISSING LINK FOR BIOMECHANIC FUNCTION? Skeletal tissues are naturally subjected to mechanical stresses resulting from locomotion movement and daily activities. Considering that mechanical forces are necessary

for functionally healthy tissues, bioreactors may have a pivotal role in the regeneration process of these tissues. As early as the 90s, researchers showed deep interest in understanding mechanical stimuli and applying them to the cells cultured in the lab by developing mechanical inductor devices: the bioreactor dynamic systems. Multiple mechanic stresses were induced by different custom-designed devices including shear stress by perfusion with minimal diffusion constraints [20,21,90,91], rotational culture [81], hydrostatic pressure [23], and compression [56]. Moreover, the use of bioreactors can improve the functionality of bioengineered constructs, accelerate production, create custom-made systems, and reduce time costs for obtaining implants for skeletal tissue applications. Despite the reduced number of commercial bioreactors, the increasing number of publications associated with bioreactor devices applied to TE strategies shows the interest and the relevance of these systems for providing in vitro mechanical stimulation (Figure 4). The mechanical stimuli can induce changes at the molecular level in skeletal and mechano-responsive t­issues. The impact of dynamic compression on chondrocyte response to mechanical forces [56] was evaluated in primary chondrocytes

TABLE 3  Summary of Biomaterials Often Used to Produce Scaffolds and Hydrogels for Skeletal Tissue Strategies Biomaterials Polymers (natural-based)

3D Template Form

Tendon

Bone

Cartilage

Polysaccharides Hyaluronic acid

Hydrogel, scaffold, growth factor carrier

[48]

[78,79]

Chitosan

Scaffold, hydrogel

[49]

[50]

Alginate

Hydrogel, encapsulation system

[49,53,54,61]

[80]

Agarose

Hydrogel

Carragenaan

Hydrogel

[56,81] [44]

[57,58]

Animal origin Collagen

Membrane, scaffold

[59,60,82]

[61–63]

[22]

Silk

Film, fibers, scaffold, microspheres

[28]

[46,64–66]

[23,46,83]

PLA/PLLA/PDLLA

Scaffold

[9,10]

[16,27]

[84]

PLGA/PGA

Scaffold, microspheres

[8,11,68]

[46]

[45,46,67]

PCL

Scaffold

[12,29]

[29]

[15]

Blends

SPCL

Scaffold

[17–19, 43,76,85]

[20,86]

Ceramics

Bioactive glass

Tubes, scaffolds, carriers

[32,87,88]

Hydroxyapatite

Scaffold

[24]

Tri-calcium phosphates

Delivery system, scaffolds

[34,77,89]

Polymers ­(synthetic)

PLA, polylactic acid; PLLA, poly l-lactic acid; PDLLA, poly d,l-lactide acid; PLGA, poly l-glycolic acid; PGA, polyglycolic acid; PCL, polycaprolactone; SPCL, starch and polycaprolactone.

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FIGURE 4  Graphic representation on the number of publications per year on the use of bioreactor devices applied for bone, cartilage, and tendon tissue engineering over the past decade (using the Web of Science®, September 2013 Thomson Reuters). The data represented for 2013 accounts only for publications referenced from January to September.

embedded within an agarose hydrogel and subjected to short-term compression. Results indicated that the provided mechanical stimuli modulated several genes and signaling pathways, which are likely to participate in the translation of mechanical forces into biochemical responses. Also, the cell responsive behavior toward mechanical stimuli may be dependent on the cell type studied. A study performed by Sheely et al. [81] showed that a rotational bioreactor proved to be beneficial for chondrocyte-based cartilage TE strategies in agarose constructs. However, bone marrow MSCs did not respond to the stimuli in the same manner, showing a ­tendency for chondrogenesis inhibition. Bioreactor devices aimed at tendon-related strategies often focus on applying cyclic mechanical loading onto tissue-engineered tendons. Moreover, in the presence of tenogenic GFs and under cyclic tensile strain, human MSCs differentiated into the tenogenic lineage, evidenced by the significant upregulation of scleraxis gene expression [9]. In a study with chitosan microchannel scaffolds, tenocytes proliferated inside the channels with production of ECM, but there was a significantly higher occupancy ratio when the constructs were under perfusion culture [21]. Bioreactor systems also provide a means for the development of large-dimensioned constructs. Culture of cellladen scaffolds in bioreactors explores an alternative for expansion and culture of autologous cells, reducing the limitations and risks associated with allogenic bone grafting (revised by Ref. [92]). But the main motivation relies on the possibility of seeding/culturing cells homogeneously throughout large constructs that can be suitable for the regeneration of critical-sized defects [19].

IV. SCALE-UP AND READY TO GO SYSTEMS The development of novel bioengineered products depends not only on the development and in vitro assessment per se but also on scale-up mechanisms that may liberate these products into the general patient populations. If it is not possible to deliver immediately, an alternative process is to store the tissue engineered construct

(TEC), stopping the process in time while maintaining the integrity of the TECs and preserving the viability of cells, and if required, cell ability to differentiate. A potential solution is cryopreservation, a well described and safe methodology to keep cells at a dormant stage for up to several decades. Also, cryopreservation would be an effective means to prepare off-the-shelf engineered tissue substitutes and preserve them to be immediately available upon request, even for autologous approaches. Despite the novelty, this preservative strategy was already considered for cartilage TE [58], with evidence for structural stability in TEC-based hydrogels [58] and the ability of encapsulated cells to maintain their proliferative and differentiation potential [58]. Porous starch-based scaffolds seeded with bone marrow cells have also been assessed for cryopreservation of TECs using standard cryopreservation methods [93]. In this study, cell viability and scaffold properties are maintained upon cryopreservation. Results also suggest that the porosity and interconnectivity of scaffolds seem to favor the retention of cellular content and cellular viability during the cryopreservation process [93]. The better understanding and control of the construct cryopreservation process will likely result in potential bioengineered products for tissue replacement and regeneration that can be preserved and immediately available upon request for patients’ needs.

V. FUTURE OUTCOMES/CHALLENGES The currently available treatments have a limited success, and TE strategies compel alternative cues and hope for the treatment of skeletal injuries to accomplish both regeneration and function reinstatement for a successful clinical ­tissue substitute. The evolution in TE will progress into the development of more complex 3D templates that combine more desirable features in a biomimetic and functional tissue substitute toward patient needs. Also, 3D templates will hold more features and merge more biochemical properties, besides the traditional physical and chemical properties, particularly in the case of

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scaffolds. The need for increasing complexity follows the increasing knowledge on cellular and tissue biology as well as on the various and combinatorial technologies for scaffold design, the timing and the sequence pattern of stimulation in the damaged site, and the understanding of inflammatory mechanisms. The strategic success lies on the delicate balance of native tissue properties addressed in a tissue substitute and its complete integration in vivo. The tuning of properties to be mimicked and combined with artificial features will potentiate the tissue substitute into clinical regenerative applications.

LIST OF ACRONYMS AND ABBREVIATIONS 3D Tridimensional AFSCs  Amniotic fluid stem cells BMP-2  Bone morphogenic protein-2 CPM  Continuous passive motion ECM Extracellular FDA  Food and Drug Administration GAGs Glycosaminoglycans GFs  Growth factors HA  Hyaluronic acid MSCs  Mesenchymal stem cells PCL Polycaprolactone PGA  Polyglycolic acid PLGA Poly l-glycolic acid PLA  Polylactic acid PLLA Poly l-lactic acid SPCL  Starch and polycaprolactone TEC  Tissue-engineered construct TE  Tissue engineering

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