81 N. TIEMPO RECOMENDADO

Integration of the engineered tissue with the wound site can be mediated by creating a scaffold that is morphologically similar to the tissue itself, thereby controlling the scaffold-tissue interface to facilitate a robust integration of surrounding progenitor cells onto, or into, the scaffold.197 This approach of starting with a similar macroscopic structure as native tissue can be thought of as a “bottom-up” approach to tissue engineering, where scientists begin with basic, bioactive materials and subsequently modify these materials to enhance the regenerative response of the host tissue. We, and others, have exploited the native properties of several proteins, including silk fibroin (SF), collagen, and fibrin, to polymerize into discrete biopolymer microthreads.198-200 SF fibers are isolated by reeling and collecting the individual fibers found in domesticated silkworm (Bombyx mori) cocoons or spider draglines (Nephila clavipes or Araneus

diadematus).200-202 Silk fibers typically are fabricated in this way with a gumming protein called sericin, which serves to strengthen and bundle the individual fibers.203,204 These microthreads

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can be organized as series of fiber-like cables in increasingly complex hierarchal structures mimicking the organization of a variety of functional tissue systems such as muscle, tendon, or ligament.21,205 Further, these scaffolds can be tailored to meet specific tissue engineering applications through changes in the manufacturing processes. By creating scaffolds from the bottom-up, there is more flexibility and control in creating a tunable, biomimetic scaffold that is instructive in facilitating and directing tissue regeneration.

Recently, our lab developed a method to control the polymerization process of fibrin to create microthreads, which have been shown to facilitate cell alignment along the longitudinal axis of the material.198 These fibrin microthreads have been implanted into a mouse model of VML injury seeded with human myoblasts, and the combination of a morphologically relevant scaffold material and highly myogenic cell line allowed for more complete endogenous muscle regeneration.76 While we observed a reduction in the deposition of scar tissue 3-4 months post- injury, histological analyses of tissue harvested at early time points suggested that the microthreads were largely degraded within 2 weeks of implantation and many of the regenerated myofibers in the wound site at later time points exhibited some degree of misalignment with respect to the native muscle tissue.76 Fibrin is rapidly degraded by proteinases, such as plasmin, within the wound site,206,207 which suggests that the rate of proteolytic degradation of fibrin microthreads needs to be decreased to be used as a scaffold for skeletal muscle regeneration.

2.5.1 Tuning Mechanical Cues

While the goal of a scaffold is not to replace the intended function of a tissue, it must be mechanically stable in its intended environment to successfully direct regeneration and degrade at an appropriate, tissue-specific rate.197,208 The mechanical properties of biopolymer microthreads vary significantly depending on the protein being considered. SF is one of the strongest protein structures that has been described, and often the mechanical and structural properties of SF are regulated at the scaffold fabrication level, such as by changing the concentration of SF within a scaffold, rather than the post-processing level, such as through chemical crosslinking, to tune the mechanical properties to that of the tissue of interest. One such modification is the removal of the gumming protein sericin through incubations in salt

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and/or detergent solutions, which lowers the tensile strength as well as improves the immunologic response in vivo.203,204

On the other hand, biopolymer microthreads constructed of proteins commonly found in

vivo, such as collagen or fibrin, are not as mechanically robust as SF. To this end, various

crosslinking methods have been described for biopolymer microthreads.198,209-211 These methods span a variety of methodologies of crosslinking, such as physical (dehydrothermal (DHT)),209,211 irradiative (ultraviolet (UV) light exposure),198,211 chemical (carbodiimide or glutaraldehyde incubation),209-212 or biochemical (transglutaminase).211 As expected, these methods increase the ultimate tensile strength (UTS), as well as the stiffness of biopolymer microthreads. Interestingly, discrete mechanical properties were developed by modifying the pH environment of the carbodiimide crosslinking reaction for fibrin microthreads.210 This suggests that, similar to saturation of transglutaminase crosslinking,213 the efficiency of carbodiimide crosslinking can be regulated both through controlling the pH environment of the crosslinking reaction as well as the total crosslinking time. These crosslinking procedures were also found to significantly increase the stiffness of biopolymer microthreads.210 Substrate stiffness is known to have a significant effect on cell behavior.214 In the case of skeletal muscle, softer substrates trigger a proliferative phenotype in myoblasts, while stiffer substrates trigger the differentiation and subsequent maturation of new myofibers.155 Such cell-specific responses to varying substrate stiffnesses highlight the need to closely match both the mechanical and biochemical environment of the scaffold with native muscle tissue to promote tissue regeneration.

2.5.2 Tuning Biochemical Cues

Biochemical cues and receptors are the primary way that cells communicate with each other and their surroundings. Altering the ECM ligands present on a material modulates the cellular response to the material. Matrix cues are known to elicit very specific cellular responses and changing the availability of different matrix molecules will alter the cell phenotype.215,216 Additionally, growth factors or cytokines can also be incorporated into scaffolds to enhance cell responses. For example, FGF2 was incorporated into fibrin microthreads to significantly increase fibroblast migration and proliferation, while the initial attachment of the fibroblasts was not affected by FGF2 incorporation.217 An advantage to using fibrin as a scaffolding material is

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that this fibrin-FGF2 system did not require any intermediate molecules, such as heparin, to facilitate growth factor binding, rather, fibrin itself has an affinity for a large array of growth factors.218 Further, a variety of peptide sequences have been identified that can be crosslinked into the fibrin matrix with Factor XIII and will serve as tethering agents to facilitate the controlled release of a specific growth factor as the fibrin scaffold is degraded.102,219-221 Other molecules such as heparin sulfate and heparin are commonly used as intermediate molecules for materials such as collagen, SF, or synthetic polymers due to their high binding efficiency to a large number of growth factors.222 These intermediaries are often tethered using common crosslinking methods such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a chemical crosslinking agent that joins primary amine groups to free carboxylic acid groups, to increase bioactivity by conjugating different growth factors or peptides to scaffolds.167,223-225

2.5.3. Tuning Cellular Microniches

Biopolymer fibers made from materials such as collagen, fibrin, or silk have inherent biochemical properties that support cell growth. In particular, fibrin is an essential part of the native provisional matrix that modulates the wound healing response and directs tissue regeneration.170,206 Unmodified biopolymer microthreads have been shown to support cell growth and outgrowth along the length of these fibers through a combination of factors including the radius of curvature of the microthreads, biochemical signaling, and integrin receptors present on the surface of the biopolymer microthreads.97,198 Additionally, MSCs maintain their multipotency while being cultured on fibrin microthreads, demonstrating that the cell microniche on fibrin microthreads is capable of supporting cells in a more plastic state.226 By altering the surface chemistry of fibrin microthreads with chemical or irradiative crosslinking, the microniche of the microthreads can be modified to direct cells to proliferate in greater number than without these surface modifications.210,217 Further, chemical crosslinking can also be used to alter the biochemical environment to induce enhanced fibroblast proliferation on braided collagen microthreads.167 By altering the structural organization of the microthreads with static axial stretching, the surface fibrillar structure of fibrin microthreads is modified to incorporate aligned topographic grooves, which direct increased cell alignment along the longitudinal axis of the microthread.194 Combined, these findings suggest that the microniche of fibrin microthreads

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is itself able to support a variety of cell functions including growth, differentiation, and higher- order functions such as complete tissue regeneration.76 Further, these scaffolds represent a dynamic platform technology that can be tuned both mechanically and biochemically to elicit specific, controlled cell functions to further guide tissue regeneration.