Mechanical cues in orofacial tissue engineering and regenerative medicine Katrien M. Brouwer, PhD1,2; Ditte M. S. Lundvig, PhD1; Esther Middelkoop, PhD2,3; Frank A. D. T. G. Wagener, PhD1; Johannes W. Von den Hoff, PhD1 1. Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands, 2. Department of Plastic, Reconstructive and Hand Surgery, Research Institute MOVE, VU University Medical Center, Amsterdam, The Netherlands, 3. Association of Dutch Burn Centers, Beverwijk, The Netherlands

Reprint requests: Reprint requests: Johannes W. Von den Hoff, Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: 10031 24 3614084; Fax: 10031 24 3540631; Email: [email protected] Manuscript received: October 30, 2014 Accepted in final form: March 11, 2015 DOI:10.1111/wrr.12283

ABSTRACT Cleft lip and palate patients suffer from functional, aesthetical, and psychosocial problems due to suboptimal regeneration of skin, mucosa, and skeletal muscle after restorative cleft surgery. The field of tissue engineering and regenerative medicine (TE/RM) aims to restore the normal physiology of tissues and organs in conditions such as birth defects or after injury. A crucial factor in cell differentiation, tissue formation, and tissue function is mechanical strain. Regardless of this, mechanical cues are not yet widely used in TE/RM. The effects of mechanical stimulation on cells are not straight-forward in vitro as cellular responses may differ with cell type and loading regime, complicating the translation to a therapeutic protocol. We here give an overview of the different types of mechanical strain that act on cells and tissues and discuss the effects on muscle, and skin and mucosa. We conclude that presently, sufficient knowledge is lacking to reproducibly implement external mechanical loading in TE/RM approaches. Mechanical cues can be applied in TE/RM by fine-tuning the stiffness and architecture of the constructs to guide the differentiation of the seeded cells or the invading surrounding cells. This may already improve the treatment of orofacial clefts and other disorders affecting soft tissues.

Orofacial clefts are the most common congenital abnormalities of the head, affecting about 1 in 600 children depending on ethnicity and geographical location.1 The etiology of orofacial clefts is not completely understood, but both genetic components as well as environmental factors are involved.2 Cleft lip and palate (CLP) patients need several surgical interventions to close the lip and restore palatal function (Figure 1). During wound repair diverse tissues are involved, including palatal skeletal muscle, skin, and mucosa. Unfortunately, CLP surgery leaves scars, which may interfere with normal development of the midface and dentition.3 Other complications include insufficient palatal muscle function which hampers swallowing, sucking, and speech.4 To prevent these complications, adjuvant therapies are necessary. Wounds normally heal by repair, which closes the defect but will lead to imperfect tissue structure. On the other end of the spectrum there is regeneration, which implicates perfect healing into the original tissue organization. Regeneration of wounds takes place in fetal tissues, but only during the early gestational period.5,6 The research field of tissue engineering and regenerative medicine (TE/RM) aims to stimulate regeneration of tissues and organs by either implanting biomaterials for in vivo regeneration, or by constructing substitutes in vitro.7,8 302

This may also be implemented in CLP treatment. A number of generally applicable requirements for a TE/RM construct must be met for optimal results: biocompatibility, biodegradability, a suitable architecture, and appropriate mechanical characteristics.9 Unfortunately, despite all the efforts, the success rate of clinical translation of TE/RM has been disappointing thus far. As tissue mechanics are crucial in the normal physiological behavior of cells, TE/RM approaches in orofacial clefting therapy may be more successful when mechanical strain is applied. In this review, we will first summarize the different types of mechanical forces normally experienced by tissues and cells. Thereafter, we will discuss studies that currently use mechanical stimulation in relevant TE/RM approaches with emphasis on muscle, skin, and mucosa as these tissues are central in CLP surgery. We will not discuss the application of mechanical strain in bone tissue engineering, as this topic has already been covered extensively elsewhere.10–12 Different methods may be required for specific tissues or organs, as the mechanical conditions in different tissues vary considerably. Applying mechanical cues in TE/RM approaches may greatly improve the outcome of surgical treatment of orofacial clefts as well as other tissue defects. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

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Figure 1. Cartoon of the soft tissues affected in CLP patients. Skin of the lip, mucosa, soft palate muscle, and bone are affected (latter not included in this review). Image courtesy of P. L. Carvajal Monroy, Department of Orthodontics and Craniofacial Biology, Dentistry, Radboudumc, Nijmegen, The Netherlands. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

TYPES OF MECHANICAL FORCES Cells constantly probe their mechanical environment and adapt their physiology accordingly. Each cell has its own mechanical niche. Depending on the tissue, cell type, and location, cells are subjected to different types and magnitudes of mechanical load (Figure 2, Table 1). Cells and tissues can be subjected to different types of external mechanical forces including (A) tensile forces, (B) compressive forces, and (C) (fluid) shear stress (Figure 2). Tensile forces increase the dimension of the tissue in the direction of the force. These are experienced by tissues such as tendons and muscles. Compressive forces reduce the dimension of the tissue in the direction of the force. For instance, body weight compresses the femoral bone. Shear stress is experienced by endothelial cells of the inner lining of the vasculature, but is also created by interstitial fluid flow in e.g., cartilage during loading. These different kinds of load cause cellular deformation or strain. Strain can differ between specific locations within the same tissue depending on the local microenvironment and composition of the extracellular matrix (ECM). The anatomical location, the macroenvironment, can also influence strain. For instance, in skin, the strain differs between locations of the body, e.g., on the scalp or the elbow. Specific lines of high tension exist (Langer’s lines) due to the orientation of the underlying collagen network.13 In addition to differences in direction, the magnitude of strain on cells also varies. For example, different muscles experience different strains, and need to produce different forces depending on their specific location and function.14 Strain is received and sensed both extra- and intracellularly. The ECM will receive most of the external strain, and depending on the interactions between the cell and the ECM, strain will be transferred to the cell. Cells can also directly pull on neighboring cells via connections such as adhesion molecules, tight junctions, gap junctions, adheC 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

rens junctions, or desmosomes. Besides strains induced by forces such as movement and growth, cells are also influenced by the mechanical properties of the surrounding ECM, and many cell types cannot survive when cultured in suspension. Stem cells have been shown to be sensitive to mechanical cues,15–25 and stem cell differentiation in vitro is influenced by the stiffness of the substrate.26 Cells probe the stiffness of the ECM and can remodel the ECM accordingly to maintain a suitable mechanical environment.27 Cells can remodel the ECM by compaction, contraction, and degradation of ECM components, but also by the deposition of new ECM. This changes matrix stiffness and composition, which also influences adjacent cells. The ECM architecture, such as shape and pore dimensions, can also modify the differentiation and responses of (stem) cells.15 In short, cell behavior is regulated by mechanical cues from the ECM (outside-in signaling), but cells also probe the mechanical properties of the ECM to remodel it to their needs (inside-out signaling). Strain is transduced intracellularly by a process called mechanotransduction, which is the translation of mechanical cues into biochemical signals. One of the key players herein are integrins, bidirectional signal transducers which deliver signals inside-out and outside-in.28 It is conceivable that TE/RM constructs should enable the binding of integrins through arginine–glycine–aspartate (RGD) peptides or other ligands to allow for signal transduction. Other key players in mechanotransduction are adhesion complexes, stretch-sensitive ion channels, and growth factor receptors.29–31 A number of excellent recent reviews have discussed this subject in depth.30,32

Figure 2. Cartoon of different strains that cells can experience. (A) Tensile strain; (B) Compressive strain; (C) Shear strain. These strains can also occur in combination. ECM, extracellular matrix. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 1. Definition of terms Term

Definition

Load Strain

All forces applied to an object Measure of deformation (change in shape or size of an object) due to an applied force Intensity of force, force per unit area Produced by applying forces from two opposing sides into an object, directed toward each other. Leading to negative strain Produced by two opposing forces that pull outward onto an object, directed in opposite directions away from each other. Leading to positive strain Displacement of layers of an object parallel to each other In one direction Identical in two directions In several directions

Stress (pressure) Compressive force Tensile force

Shear force Unidirectional Equibiaxial Multidirectional/multiaxial

Magnitude (Unit) F (N) e (unitless) r or P (N/m2 or Pa) F (N) F (N)

F (N) N/A N/A N/A

N/A, not applicable.

For TE/RM, several options for the application of load to constructs are available. Load can be applied in vitro in the form of stretch, fluid shear stress, and compression: high-tech bioreactors allow protocols with varying duration, interval, flow, and strain to match the specific needs of the target cells and tissues.33 In the following paragraphs, we will discuss how mechanical stimulation might be useful in TE/RM of skeletal muscle, and skin and mucosa. These soft tissues are all affected in CLP patients by cleft surgery, but also in other conditions such as trauma, tumour resection, or in other congenital defects. Specific mechanical cues may increase the quality of soft tissue constructs, and hence their clinical application.

SKELETAL MUSCLE AND MECHANICAL STRAIN Skeletal muscle is a clear example of a soft tissue that is influenced by mechanical forces. It consists of muscle fibers aligned parallel to the main lines of tension. Tension during muscle development causes an increase in myofiber length and myofibril number, and increases protein accumulation in later phases.34 In the adult situation, muscle cells turn hypertrophic in response to exercise, whereas atrophy occurs when muscle is not stimulated for longer periods of time. This indicates the importance of mechanical stimulation in the physiological function of muscle tissue and in the development of TE/RM muscle constructs. To prepare a functional muscle construct, muscle cells should proliferate and differentiate into aligned, cross-striated myofibers that produce physiological forces. In general, cells can respond to mechanical stimulation by changing their proliferation rate and differentiation status.35–40 Applying mechanical strain stimulates satellite cells—the normally quiescent muscle stem cells—to become activated.41 This might be caused by the release of hepatocyte growth factor from the ECM by stretching.42,43 The proliferation of avian myoblasts and murine C2C12 myoblasts is stimulated by mechanical strain.36,39,44,45 Muscle cell differentiation is also 304

enhanced by mechanical stimulation.39,46,47 In contrast to these findings, cyclic mechanical strain can also reduce the differentiation of C2C12 mouse myoblasts, adult bovine myoblasts, and murine muscle progenitor cells.36,44,45,48 These differences might be related to the applied strain regimen. Cells can be stretched unidirectionally or multi/(equi)biaxially. Stretching protocols can be used to create ECM alignment in constructs, which, vice versa, influences cell metabolism.49,50 This knowledge may be used to recreate the original ECM orientation of the tissue of interest. It should be kept in mind that cells can align differently depending on the applied strain. Cells have been shown to align parallel to the strain direction under static strain,39,51,52 and perpendicular under dynamic or cyclic strain.51,52 The latter is called stress shielding, and is hypothesised to be caused by the development of micro-ripples perpendicular to the strain.53,54 These observations are highly relevant for tissue regeneration as collagen is deposited primarily along the cells’ main axis in vivo, and constructs with an orientation like the original tissue may provide the best basis for regeneration. Uniaxial stretching of myoblasts increases the differentiation and alignment compared with equibiaxial stretching.55 Multiaxial stretch was shown to increase the phosphorylation of ribosomal S6 kinase, involved in muscle hypertrophy.56 The orientation, force and strength of strain can thus influence the differentiation of muscle cells. Another hurdle is that it is difficult to determine the exact strain experienced by muscle cells in vivo. To complicate matters further, the observed responses in vitro may also be caused by changes in nutrient diffusion47 and contact inhibition.39 These stretch-dependent outcomes underscore the need of well-established protocols before clinical translation.

MECHANICAL STRAIN IN TE/RM OF OROFACIAL MUSCLE In CLP patients, the major muscle of the soft palate (m. levator veli palatini, LVP) needs to be reconstructed for proper velopharyngeal functioning and normal speech development.57 A method to reconstruct this muscle is the C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

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implantation of a (cellularized) scaffold in the palatum. Cells may experience different levels of strain when cultured in vitro in a two-dimensional or in a more biomimetic three-dimensional (3D) model.33 An often used 3D model for tissue-engineered muscle is the production of myofibers in ECM-based hydrogels. These constructs allow assessment of the effects of strain on muscle cell proliferation and differentiation. The molecular responses to movement/exercise in such a model58 were comparable to the in vivo situation in terms of growth factor synthesis and force production.47,59–61 The stiffness of a substrate can determine whether cells will proliferate or differentiate.62 For in vivo use, hydrogels may be less suitable due to their limited mechanical stiffness and force production.63 The mechanical stiffness of hydrogels can be improved by chemical crosslinking,64 but the maximum force they can produce is still about seven times lower than native adult skeletal muscle.63 Using unstrained decellularized muscle scaffolds seeded with C2C12 mouse myoblasts, in vitro, only low forces were produced.65 Comparable constructs seeded with human satellite cells and cyclically strained in a bioreactor, gave a contractile response in vivo of 1% of that of native muscle. In contrast, statically cultured muscle constructs did not generate any contractions.66 This indicates that mechanical preconditioning can increase the produced force. However, the implantation of these constructs in a rat limb muscle defect increased the functional capacity in only about 50% of the cases.67 This may be related to a stronger immune reaction in the “negative responders.”68 Another issue is the limited myofiber survival after implantation of muscle constructs.68,69 These experiments show that in vivo implantation does not readily lead to long-lasting improvement in muscle function. Moreover, several studies have shown a decrease in regenerative capacity in vivo after in vitro culture of muscle cells,70–72 which might be related to dedifferentiation.73 An alternative approach would be to skip the in vitro culturing step and to apply in vivo TE. Then, the cells are isolated, seeded into an appropriate scaffold, and implanted directly into the defect.74 This construct will then be exposed to a physiological level of mechanical force, avoiding the time-consuming culture step and complicated regulatory issues. However, obtaining enough cells from biopsies without culturing may be impossible. A totally different view on the effect of mechanical load on muscle regeneration emerged from a study by Corona et al.75 They observed that decellularized muscle ECM was unable to induce muscle regeneration, but still muscle function had improved significantly. A possible explanation for this could be that the construct protected the remaining muscle from overload.75–77 Although TE/RM research has focused rather intensively on skeletal muscle, this mainly concerns muscles of the limbs and trunk. There are, however, significant differences between these muscles and the muscles of the head (reviewed in Carvajal Monroy et al.4, probably related to their different embryonic origin,78,79 which gives rise to architectural and cellular differences. Craniofacial muscles regenerate slower, and seem to produce more fibrotic tissue after injury than limb muscles.4 In vitro, the satellite cells of the masseter muscle (one of the muscles of mastication) proliferate more and differentiate later.4 Muscle fiber types also differ between muscles, and the LVP in C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

healthy individuals contains a larger proportion of slow fibers, whereas that of CLP patients has a higher proportion of fast fibers.80–82 Stretching seems to guide the type of muscle fibers formed in culture, as MyHC-2b, a fasttwitch subtype, is up-regulated after rapid stretching of C2C12 cells, whereas slow stretching induces the slowtwitch MyHC-2a and MyHC-1.83 Fast muscle fibers generally contain less satellite cells,84,85 and these cells proliferate less in vitro.78 Moreover, mechanical strain stimulates protein synthesis and fiber growth in glycolytic fast fibers less than both slow and fast oxidative fibers from rat soleus muscle.86 During exercise, protein synthesis first decreases, to increase again after a lag phase.87–90 This is specifically the case in muscles with a high proportion of fast glycolytic fibers,87 and thus of particular relevance for CLP patients. These data indicate that specialized protocols are needed for the stimulation of proliferation and differentiation for orofacial muscle cells. Muscle regeneration in the soft palate of CLP patients may be limited due to fibrosis, reduced differentiation of myoblasts, and reduced protein synthesis. All muscles have a different force production, which may ask for “customized” protocols. For instance, jaw muscle needs to be able to produce more force, whereas muscles in the soft palate, which are used during speech, sucking, and swallowing, need to have a better endurance. A slow stretch protocol stimulating slow, enduring muscle fiber formation may, therefore, be most appropriate for CLP muscle TE/RM. However, the application of TE/RM in the orofacial region is still in its infancy, and specific animal models such as a recently developed palatal wound model91 are required to evaluate TE/RM approaches for the muscles in the orofacial region.

SKIN AND MUCOSA AND MECHANICAL STRAIN Mechanical factors are involved in the physiology of normal skin and mucosa. Its elasticity, for instance, protects skin from rupture during contact. This elasticity is derived from the basket-weave orientation of the ECM fibers collagen and elastin.92,93 During wound healing, several mechanical issues are involved, including the wounding itself, but also the pulling by myofibroblasts during wound contraction. Mechanical properties are also altered by granulation tissue formation and edema.94 Suturing of a wound leads to mechanical strain, especially when a larger tissue defect is present. Moreover, wounds over the joints heal with more scar formation,29 showing that mechanical tension increases scarring.95 Normal skin and scar tissue have different mechanical characteristics.96–99 In scars, collagen type I has a more parallel orientation than in normal skin, which reduces compliance at low loads and resistance to failure.97 Thus, constructs should guide proper collagen deposition as tissue stiffness may compromise function. As strain contributes to (hypertrophic) scar formation, methods have been developed to reduce strain on wounds. Stress-shielding devices that reduce the mechanical strain have been developed to diminish scarring. In studies on porcine skin wounds, the scar area was strongly reduced in stress-shielded wounds compared with control and highly stressed wounds.100 The beneficial effect of stress305

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shielding was also confirmed in a human study.100 Another method is the use of negative-pressure wound therapy, in which a vacuum is created over the wound. This decreases wound surface area and enhances healing rates,101–103 possibly through enhanced granulation tissue formation, angiogenesis, cellular proliferation, and myofibroblast differentiation.101,104 In contrast, pressure therapy is also used to reduce scar formation. Pressure garments have been found to improve scars with respect to maturation and hypertrophy. They seem to reduce collagen synthesis by limiting the blood supply, and to help realignment of collagen fiber bundles (reviewed in Macintyre and Baird105). Scar formation can thus be reduced by an initial reduction of (tensile) stress on the wound (stress-shielding devices), whereas later on pressure therapy limits hypertrophic scarring. Comparing these two methods, the strain direction seems to be different, as stress shielding reduces tensile strain parallel to the skin surface, whereas negativepressure therapy and pressure garments strain the skin perpendicular to the surface.

MECHANICAL STRAIN IN TE/RM OF OROFACIAL SKIN AND MUCOSA The structure and composition, but also the mechanics of skin and mucosa vary for different locations in the body. This may have implications for TE/RM approaches of specific locations such as the orofacial region in CLP patients. Skin has a different mechanical orientation (Langer’s lines) all over the body, and it differs locally in thickness and structure. Skin in the lip region, for instance, is rather thin, as opposed to skin on the palms of the hands. Skin around the lips is also well perfused, which may be beneficial for healing. The healing of oral mucosa is generally faster and leads to less scar formation.106 This may be caused by healing-promoting factors in the saliva,107 but also by an inherent difference in phenotype between oral and skin tissue and fibroblasts, with respect to resolution of inflammation, ECM remodeling and ECM expression.108,109 This latter is especially important, as a dermal construct should stimulate fibroblast proliferation and differentiation, but conversely, the balance between cell number and ECM (production) is crucial in skin regeneration as hypertrophic scarring involves too many myofibroblasts and too much ECM. Mechanical loading of wounds causes hypertrophic scarring in mice,110 and both in vitro and in vivo studies have shown that static as well as cyclic strain increase myofibroblast formation,111 proliferation,37,112,113 and differentiation (the expression of several ECM genes including a smooth muscle actin [aSMA]).37,112–114 Strain was also found to reduce myofibroblast apoptosis,110,115 whereas its release increased apoptosis.116 Others have shown that aSMA is down-regulated after cyclic equibiaxial strain in vitro,117 and these differences may result from different strain profiles.118 Strain direction and donor age also affect responses regarding cellular morphology, proliferation, and collagen production.119,120 Finally, in lung fibroblasts differentiation is reduced after cyclic strain,121 in contrast to dermal fibroblasts that generally showed increased differentiation.37,112,113 In gingival fibroblasts, mechanical strain leads to proliferative and antiapoptotic stimuli,122 whereas others confirmed the induction of pro306

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liferation but did not observe increased expression of myofibroblast markers.123 Cell type is thus also important in responses to mechanical strain, and results should, therefore, specifically be defined for cells from the orofacial region. In the epidermis, the specific architecture of the rete ridges provides a mechanical niche for keratinocytes. This knowledge may be used in TE/RM, as in constructs with an architecture comparable to this rete ridge structure the epithelialization rate was higher than in constructs with a flat surface, and the expression of genes related to proliferation and migration was increased in keratinocytes.124–126 Mechanical strain has further been found to increase the proliferation of keratinocytes by both static and cyclic stretch,38,40 whereas differentiation was down-regulated by static stretch.40 Keratinocyte proliferation may be beneficial in the closure of wounds but can also cause a thickened epidermis as observed in hypertrophic scars and psoriasis.127 Normal skin and mucosal ECM fibers are present in a random, basket-weave orientation, although alignment of collagen fibers has been observed in specific locations.128 Scar tissue usually has more parallel collagen fibers than normal tissue. In clinical wound closure, skin is sometimes stretched to close larger defects, leading to a more parallel alignment of collagen and elastin bundles.129 The effects on the quality of the resulting skin are not exactly known. Mechanical strain can thus influence the orientation and density of (newly deposited) fibers within a construct, and therewith skin quality. This may in turn modulate the cells, as for instance the orientation or nanotopography of the substrate has been found to influence fibroblast migration.128 The effects of a unidirectional orientation or a more random orientation of ECM fibers both in vitro as well as in vivo should, therefore, be investigated. In vivo, faster vascularization and myofibroblast ingrowth was observed in collagen scaffolds implanted in the mucosa covering the hard palate compared with scaffolds implanted in the skin of rats. Scaffolds implanted in the loose skin on the back of the rats showed more inflammation compared with scaffolds in the palate (which lies over bone) or in the skin on the skull. This might be caused by the higher level of mechanical stimuli in the back skin.130 These differences indicate the importance to perform studies specifically for the orofacial region. Another method to implement strain in TE/RM for skin is by controlling scaffold stiffness. Higher scaffold stiffness accelerates angiogenesis, decreases wound contraction, and improves dermal regeneration.131 As the application of mechanical strain during in vitro culture can also improve the (experimental or surgical) handling properties of the constructs by increasing ECM production,37,132 these are all beneficial effects of a higher construct strength.

CONCLUSION AND FUTURE DIRECTIONS The surgical treatment of CLP is still suboptimal, and TE/ RM strategies could be useful to improve regeneration of the tissues involved. As mechanical strain is important for the development and maintenance of healthy tissues, TE/ RM constructs may also be enhanced by applying strain. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

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First, it is essential to define the desired aim of mechanical stimulation for regeneration. During production of a skeletal muscle construct, the aim is to stimulate cellular proliferation and differentiation, whereas when implanted the construct should experience and produce physiological strains. Although the reported optimal strain parameters for well-differentiated muscle constructs vary, the application of strain by stretching is probably the most physiological form of mechanical stimulation. For CLP muscle TE/ RM, unidirectional strain using a slow stretch protocol may be most appropriate as this will stimulate the development of slow twitch, (endurance) muscle fibers, which are present in normal palatal muscle. For use in vivo, the stiffness of constructs used for muscle TE/RM should be increased by the use of other materials than hydrogels. Vascularization and innervation should be stimulated to optimize myofiber survival and force production,73 using for instance growth factors. Although in vitro mechanical strain can improve the orientation and strength of muscle constructs, the results are not yet optimal. In vivo TE/RM by the implantation of an acellular scaffold with chemotactic agents for (stem) cells seems to be promising. Alternatively, cell-seeded scaffolds could be implanted after which the cells are stimulated by the physiological load on the construct. In vivo TE/RM with acellular constructs would also ease the extensive regulatory issues and high costs associated with culturing cells for clinical application. Mechanical factors are crucial for the normal physiology of skin and mucosa, but forces on a wound may also increase the risk of (hypertrophic) scarring. Scar formation can be diminished by limiting these forces, which is already clinically applied in pressure therapy. For CLP patients, the most obvious method is to prevent tight suturing and to use constructs that can withstand the forces on the wound. The response of skin cells to mechanical strain in vitro varies considerably depending on strain type, strain direction and cell type and origin, which as yet precludes the establishment of solid protocols. It is evident, however, that the application of mechanical strain in vitro can strengthen constructs before implantation. Moreover, constructs with an appropriate ECM composition and geometry, in combination with a suitable stiffness, may already improve regeneration without external mechanical stimulation. Concluding, there are several opportunities to implement mechanical cues into TE/RM approaches for CLP and other soft tissue disorders. Currently, the most feasible approach to provide mechanical cues to cells is by optimizing scaffold architecture and stiffness, and not by in vitro mechanical stimulation. The effects of dynamic vs. static mechanical strain on cellular differentiation and orientation should be further elucidated as the present results are contradictory. We also conclude that more research should be performed to optimize the parameters for in vitro loading of soft tissue constructs to further improve translation to the patient. Source of Funding: This work was supported by the Dutch Burns Foundation project 09.110, TASENE (NWO/ SIDA/COSTECH W02.29.101), and the European Commission (FP7-HEALTH-2011-1, Grant Agreement no. 279024), project EuroSkinGraft. The authors declare no conflicts of interest for this work. C 2015 by the Wound Healing Society Wound Rep Reg (2015) 23 302–311 V

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