Evaluation of Silk as a Scaffold for Musculoskeletal Regeneration – the Path from the Laboratory to Clinical Trials

5.27 Evaluation of Silk as a Scaffold for Musculoskeletal Regeneration – the Path from the Laboratory to Clinical Trials O Hakimi, Oxford University Institute of Musculoskeletal Sciences, Oxford, UK F Vollrath and AJ Carr, University of Oxford, Oxford, UK © 2011 Elsevier B.V. All rights reserved.

5.27.1 5.27.2 5.27.2.1 5.27.2.2 5.27.3 5.27.3.1 5.27.3.2 5.27.3.3 5.27.3.4 5.27.3.5 5.27.4

Introduction Common Types of Silk Scaffolds Scaffolds Based on Native Fibers Scaffolds Based on Regenerated Silk A Review of Studies of Silk Scaffolds for Musculoskeletal Tissue Engineering Bone Muscles Cartilage Tendons Ligaments and Joints An Evaluation of Silk as a Scaffold for Musculoskeletal

Repair – in the Context of Medical Device Regulations 5.27.4.1 History as a Suture 5.27.4.2 Chemical Characterization of Silk 5.27.4.3 Toxicology 5.27.4.4 Local Effect after Implantation 5.27.5 Summary Acknowledgments References Relevant Websites

Glossary Bombyx mori The domesticated silkworm, producer of the best-characterized silk. Commercial silk sutures (such as Mersilk) are made from B. mori fibers, as well as the vast majority of experimental cell scaffolds. degumming A process to remove the sericin gum from silk fibers. The standard is typically boiling the silk fibers in alkali soapy water.

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fibroin The chief protein component of silk fibers. A repetitive, fibrous, hydrophobic protein rich in β sheets. sericin A family of serine-rich glycoproteins secreted by silkworms as the glue coating of silk.

5.27.1 Introduction Numerous conditions of the musculoskeletal system can be solved or relieved by the use of engineered tissues. For example, loss of cartilage and tendon (due to trauma, wear, or aging) are common and debilitating conditions, where the defect, often combined with poor-quality tissue, causes pain and limited movement. Relevant studies have shown that the implantation of a graft or scaffold can potentially enhance and speed up the repair. Similarly, in bones, despite the inherent ability of this tissue to heal, large lesions may arise as a result of tumors or osteomyelitis and are associated with greater risk of nonunion. Tissue engineering has been proposed as an efficient means of improving regeneration in these situations [1, 2]. To repair or replace musculoskeletal tissues, a few groups of materials, including synthetic degradable polymers (such as the poly (α-hydroxy)acids) and natural polymers such as collagen, chitin, and silk, have been proposed to act as scaffolds [3]. While absorbable synthetic polymers are relatively safe and allow scientists excellent control of scaffold properties, natural polymers are characterized by superior interaction with cells. Among these natural polymers, silk is outstanding in its versatility, slow degradation rate, and superb mechanical properties. This combination of properties, discussed in more detail below, has positioned silk as a promising candidate material for skeletal tissue engineering [4–7]. A number of studies have investigated four major aspects of silk as a biomaterial: (1) mechanical suitability; (2) formation of a scaffold that mimics the relevant tissue; (3) the ability of silk to support the attachment, growth, and differentiation of cells forming the musculoskeletal system as well as their progenitors, mesenchymal stem cells (MSCs); and (4) the use of animal models to

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Idea development Specific need for an implant

Scaffold fabrication

Design of a test system to evaluate feasibility of concept

Preclinical studies Chemical characterization of material In vitro cytotoxicty Product safety: toxicology, genotoxicity, and carcinogennicity Interaction with blood Local effect after implantation Identification and quantification of degradation products Toxicology of degradation products Tests for irritation and hypersensitivity

Clinical trials Initial trial in a small group of patients: comparison to an existing implant

Larger trial to establish efficacy and safety

Figure 1 A brief overview of the process for translating an implant idea into a clinical treatment. Special emphasis is on the numerous requirements at the preclinical stage, which can also be a summary of past in vitro and animal studies [8–10].

evaluate the integration and performance of different silk scaffolds in vivo. As is shown below, the majority of these studies yielded promising and positive results. In the light of so many positive preclinical studies, and the existing Food and Drug Administration (FDA) approval of silk sutures, it is surprising that there is very little information available on current clinical trials of silk implants. Thus, the chief aim of this article is to evaluate the feasibility of translating silk studies in laboratory and animal models to clinical trials (Figure 1). The first part of this review briefly summarizes the relevant past studies of silk for skeletal tissue engineering. The second part evaluates findings from past studies in the context of medical implant assessment and safety, and highlight aspects that merit further research.

5.27.2 Common Types of Silk Scaffolds There are two main types of silk scaffolds typically used for musculoskeletal tissue engineering: scaffolds derived from native silk fibers [11, 12], and scaffolds formed from regenerated silk [13]. Occasionally, investigators have coated native fibers with regenerated silk, thus combining these two scaffold types in one [14]. The predominant silk used is Bombyx mori, from the domesticated silkworm, although a number of studies have used the wild silkworm Antheraea and a few species of spider silk.

5.27.2.1

Scaffolds Based on Native Fibers

Scaffolds constructed from native fibers are typically cleaned from their coating (degummed) by the conventional degumming method of boiling the silk with soap alkali solution. Recently, the use of proteases for degumming has also been reported [15–17]. The degumming is described as an essential step to remove the sericins, the glycoprotein coat of silkworm silks. Sericins have been implicated in inflammation and allergic responses [4, 18], which will be discussed in more details below. Once sericin is removed, the major component of the clean fibers is fibroin. However, there is currently no standard of cleanliness, that is, fibroin purity. Once silk fibers have been degummed, they are braided [12], woven, or combined into a nonwoven construct [6, 19] to create a scaffold. The obvious advantage of using native fibers is retention of their excellent mechanical properties, and, even after degumming, silk retains excellent strength and elasticity [20]. Moreover, the morphology and ultrastructure of native silk fibers is strikingly similar to fibrillar tissues such as tendon, suggesting they may be useful as replacement grafts (see Figure 2). A key disadvantage of using native degummed fibers lies in the lack of precise knowledge of all the chemical components of the fiber. Not only is the extent of sericin removal unknown, but there may also be additional as-yet uncharacterized components in the fiber [23]. Moreover, native fibers can be highly irregular, and may present variation between populations, individual worms, and even within the same fiber [24].

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Figure 2 Scanning electron micrographs of collagen fibrils in rat Achilles tendon [21] [a] and Antheraea pernyi (wild silkworm) silk [22] Image reproduced with permission. (b,], showing remarkably similar, very dense, and well-aligned ultrastructure.

5.27.2.2

Scaffolds Based on Regenerated Silk

Regenerated silk scaffolds are usually prepared by dissolving the degummed fibers in chaotropic agents (such as lithium bromide), thus denaturing the fibroin protein. The solution is then dialyzed in water, and scaffolds can be prepared from the aqueous solution by casting, freeze-drying, or mixing with additional components [13, 25]. The fibroin solution can also be used to coat other scaffolds [26, 27]. The advantage of using regenerated silk is the good control this process gives over the scaffold properties. It is possible to modify the density, shape, porosity, degradation profile, and tensile properties of the material [13, 28]. The regenerated silk solution can also be used as part of a composite material [29–31]. However, a major disadvantage could be the potential of high β-sheet content of the newly formed fibroin-based material [32], which could be linked to amyloid formation in the tissue [33]. Moreover, using harsh solvents during silk processing may worsen cell interaction with silks due to the persistence of residuals [34]. As is shown below, the significant structural differences between native and regenerated silks [35] are also apparent in their interactions with biological tissues and fluids, and require their separate assessment for efficacy and safety.

5.27.3 A Review of Studies of Silk Scaffolds for Musculoskeletal Tissue Engineering The following paragraphs will summarize work done on a variety of silk scaffolds to support the formation of musculoskeletal tissue.

5.27.3.1

Bone

A large range of silk scaffolds has been produced to support bone formation in vitro and in vivo. Most of the scaffolds were based on regenerated silk, although one study explored degummed and nondegummed silk cloth [36]. Fabricated scaffolds included silk films [37, 38], fibroin hydrogels [25], disks or porous three-dimensional (3D) constructs [39, 40], synthetic polymers coated with fibroin [26], and composite porous structures [30, 41]. In many of the studies, control of scaffold size, porosity, and chemistry were demonstrated as a means to optimize scaffold performance, for example, by improving the degradation time [37, 39]. Moreover, these studies have demonstrated that both osteoblasts and human MSCs can easily attach, grow, differentiate, and form neo-bone on silk scaffolds [25, 26, 37, 38, 42]. At least in vitro, regenerated fibroin has been shown to enhance cell attachment and proliferation, and, under certain conditions, has performed better than collagen and poly-L-lactic acid (PLLA) scaffolds [26, 38, 40]. Moreover, some silk scaffolds were shown to possess mechanical properties similar to bone [30]. In vivo, silk scaffolds have been implanted in rabbit, mouse, and sheep models, and the authors reported good performance of these implants with some degradation, some bone formation and remodeling, and, importantly, no inflammation [25, 30, 40, 41]. Overall, these results show excellent proof of concept for silk as a bone scaffold.

5.27.3.2

Muscles

Muscle tissue engineering is motivated by the need to carry out basic research in vitro, as well as for applications in prosthetics and robotics. Very little work can be found on silk in this context, but one purely mechanical study by Agnarsson et al. [44] has linked the shrinking of silk in water or under certain humidity (a phenomenon named supercontraction [43]) to its ability to act as an artificial muscle. By responding to cyclic changes in humidity, silk fibers have shown large displacements, and could theoretically be used to mimic muscle contraction. This study discussed silk as a novel biomimetic muscle, but did not propose a practical biomedical application [44].

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% Viable cells on sutures compared to tissue plastic control

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0 Mersilk

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PDSII

Ethibond

Suture type Figure 3 Tendon-derived fibroblasts survival on different suture material over 96 h in vitro. Spidrex, an Antheraea pernyi-based suture, performed better than existing the Bombyx silk suture, Mersilk. Bars represent standard deviation. (Unpublished, Hakimi and Carr, 2009)

5.27.3.3

Cartilage

In a similar fashion to bone, a large body of work has demonstrated the possibilities and potential of silk scaffolds to support cartilage tissue engineering. A versatile range of silk scaffolds, including injectable matrix [45], films [46], and porous 3D constructs [28, 46–48], has been produced to support chondrogenesis. Composite scaffolds have combined fibroin with RGD peptides [49] and hyaluronan [50]. A few studies used native spider silk fibers from Nephila edulis [6] and Araneus diadematus [14] in the form of nonwoven meshes. These studies have shown that silk in its various forms could support the attachment and growth of chondrocytes [6, 14, 28, 48] and MSCs directed toward chondrogenesis [46, 47, 49, 50], performing better then chitosan scaffolds [47] and tissue-culture plastic [46]. The performance of the scaffolds was evaluated by measuring cell density and growth in vitro, extracellular matrix (ECM) synthesis, and spatial arrangement of cells. Taken together, these studies established silk as a good candidate for cartilage regeneration.

5.27.3.4

Tendons

Compared to bone and cartilage, fewer studies have investigated the ability of silk to support tendon repair. Here too, both regenerated silk [51] and native degummed fibers have been studied [12]. In vitro, tenoctye growth on Arg–Gly–Asp (RGD)­ modified silk films was superior to that on tissue-culture plastic [51]. Unpublished data from our lab show excellent cell attachment and survival of tendon-derived fibroblasts on A. pernyi-based sutures, Spidrex (see Figure 3). In vivo, the implantation of braided A. pernyi silk to repair Achilles tendons in rabbits resulted in improved collagen alignment of the repair, although the authors reported some inflammation at week 2 and degradation of the fibers into fragments [12].

5.27.3.5

Ligaments and Joints

Many of the studies on silk for joint and ligament regeneration focused on native fibers, albeit often modified. Fibers were twisted into a wire rope, knitted, or simply bunched together, before they were decorated with RGD peptides [52], and embedded in collagen coating [11], in gelatin [53], or in regenerated fibroin [54]. In one study, the fibers were used to provide mechanical support by reinforcing a hydrogel [55]. Typically, scaffolds were loaded with MSCs prior to in vitro experimentation or in vivo implantation [11, 54]. Reported results from in vivo implantation in rabbit [11, 54] and rat models [53] were incredibly positive. The authors observed that little or no inflammation occurred around degummed and coated fibers. This was despite a very lengthy implantation period of 360 days and is somewhat unexpected. Chen et al. [11] also reported no measurable loss of scaffold during the experiment. To summarize, experience with silk scaffolds for musculoskeletal repair has been highly encouraging, with both in vitro and in vivo results indicating excellent proof of the concept that silk is not only exceptional mechanically but is also highly appropriate for supporting the cells in forming a neo-tissue. The following section will examine evidence relevant to the safety of the different silk scaffolds as novel implants.

5.27.4 An Evaluation of Silk as a Scaffold for Musculoskeletal Repair – in the Context of Medical Device Regulations 5.27.4.1

History as a Suture

B. mori fibers have a long history as sutures, dating from as early as the nineteenth century, and are still in use today in the form of braided yarns coated with beeswax or silicone. Silk suture is often considered as standard due to its superior handling characteristics.

Evaluation of Silk as a Scaffold for Musculoskeletal Regeneration – the Path from the Laboratory to Clinical Trials

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It is officially classified as a nonabsorbable suture, although it is known to degrade by proteolysis and lose its tensile strength within a year of implantation [56]. Silk sutures have also been linked to allergy and foreign body response. In comparative studies of sutures implanted in animal models and humans, silk sutures have usually received a higher inflammation and granuloma score than synthetic polymer sutures [57–60], although, in a few cases, silk has scored the same [61] or better [62]. Some severe reactions to silk sutures have been reported after thyroid surgery [63] and hernia repair [64]. One study has also shown that silk suture is carcinogenic in rats [65], but a comparative study showed no difference in tumor yield between silk and other sutures [66]. In a few rare case studies, adverse responses were reported as late as 7 years after subcutaneous implantation with severe foreign-body response to the silk sutures, chronic areas of reaction around the sutures, necrosis, and granuloma [67]. Foreign-body reaction to an implanted material is thought to be tightly linked to its surface properties, including both chemistry and morphology [68]. As the commercial B. mori silk suture is coated with wax/silicone and dyed black, it is difficult to conclude if fibroin itself is responsible for cases of severe inflammation. Conversely, one study linked these severe reactions to silk allergy, which can be anticipated by an intradermal skin test [63]. Thus, the history of silk as a suture, despite being extensive and widely studied, does not always provide conclusive data about the safety of using regenerated silk or native fibers, and, therefore, should not be taken as definitive for all silk scaffolds.

5.27.4.2

Chemical Characterization of Silk

Regulatory bodies require chemical characterization of implants [8]. Native mulberry silkworm silk fibers are known to be primarily composed of the fibrous protein, fibroin, which is well characterized in terms of its sequence and secondary structure [69–72]. The structure of fibroin has also been investigated in its regenerated form [73, 74]. Work has also been done to characterize the glycoprotein family sericin, which forms the gum-like coating of silk fibers [75, 76]. However, there may still be other, unknown components in silk which are of importance in the clinical setting. A few studies isolated and characterized additional molecules in native silk fibers and hypothesized that these may take part in pigmentation [77], or defense against predators [23, 78]. Spider silks, moreover, can contain a wide range of surprising compounds, ranging from glycoproteins to neurotransmitters [79, 80]. Thus, a complete characterization of all silk-fiber components from different populations of silkworms and spiders remains an important challenge.

5.27.4.3

Toxicology

Preclinical studies of a new implant should prove that it is safe in both the short and the long term, including after its degradation and removal. According to the classification of the implant, it may be necessary to test parameters such as genotoxicity, carcino­ genicity, pyrogenicity, interaction with blood (hemocompatibility), irritation, and hypersensitivity. Toxicity may be tested both in vitro and in vivo, and tests in rabbits and rats are the standard [8, 10]. Most in vitro studies provide an excellent profile for silk as a biomaterial. Many other studies have demonstrated that degummed and regenerated silks, especially when coated with matrix proteins such as collagen, can generally support cell growth in vitro [4, 13, 27, 29, 34, 81]. However, only a handful of studies quantitatively assessed the compatibility of silk with human cell cultures. One study has demonstrated that silk sutures coated with collagen had no toxic effect on MSCs over 12 days in culture [82]. Standard degummed, uncoated A. pernyi has been shown to have strong in vitro toxicity, but enzymatic degumming removed this effect [17]. Films from regenerated silkworm silks of B. mori and A. mylitta were also compatible with fibroblast cell cultures grown over 7 days [83]. The degradation products of regenerated silk fibroin were also nontoxic to human neuronal cells [84]. However, soluble factors from braided silk suture have been reported to exert a considerable effect on macrophage adhesion [85]. The hemocompatibility of silk has also been studied with different results for silk sutures, native fibers, and regenerated scaffolds. In comparison to other commonly used sutures, silk sutures had a high thrombogenic activity [86]. However, when comparing native and regenerated silks, Santin et al. [87] found significant differences, where regenerated B. mori fibroin films were relatively inert to human plasma compared to native fibers, to which plasma had strong affinity. The authors have suggested an explanation for this difference, where the hydrophobicity as well as the possible presence of specific domains on the surface of the native B. mori silk can prompt fibrinogen binding. Unlike regenerated silk films, which were hypothesized to have a more hydrophilic and less ordered surface [87]. This is in agreement with another study, which has shown regenerated B. mori silk to have excellent patency as a vascular graft with anti-thrombogenic activities [88]. Moreover, other studies have shown that it is possible to optimize the interaction of proteins with the surface of regenerated silk by immobilizing an anticoagulant factor [89] or varying processing conditions [74]. Very little information is available about the genotoxicity and carcinogenicity of silks. At least one study has shown that regenerated B. mori silk had no evident genotoxicity [90]. In one communication, a commercially available silk suture has been suggested to exert a co-carcinogenic effect [65]. Carcinogenicity around the site of polymer implantation is linked to the implant’s surface area, surface smoothness, and degradation rate, with slower degrading polymers more likely to induce tumors [10]. Thus, more work may be needed to ensure that silks do not increase sarcoma incidence. Finally, there are numerous reports about allergenic reactions to silks and their derivatives, such as silk waste used for the production of heavy fabrics or filling material for bed quilts [91]. A few components of silk have been suggested as the allergens,

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including peptides of low molecular weight speculated to be sericins [92] and the B. mori fibroin protein itself, both heavy and light chains [93]. There have also been suggestions that the allergens do not originate from the silk yarns, but are external contaminants from the processing [91], or that patients were sensitized by contact with antigens of moths or butterflies rather than silk products [94]. These studies do not provide a conclusive indication as to which part(s) of the silk are likely to be the cause for reported allergies. To summarize, cytotoxicity studies have clearly demonstrated not only good interaction between silk scaffolds and human cells, but also the importance of distinguishing between regenerated and native silks, as well as existing commercial silk sutures. Especially in terms of blood compatibility, differences between regenerated and native fibers should influence the choice of their application. In addition, more published quantitative and long-term data could strengthen the case for silk as a biocompatible material. Finally, the allergenic nature of silk is still unclear, and must be resolved prior to clinical studies.

5.27.4.4

Local Effect after Implantation

In vivo implantation tests are critical for evaluating the localized effect of a biomaterial on the tissue. Histopathology of the active tissue around the implant and more distant tissues in a rabbit or a mouse model is considered the acceptable standard. However, unlike drugs, the reaction to a biomaterial is considered not only dose dependent but also time dependent, and regulating bodies require a full evaluation of immune response over the life of the implant [8]. Taking into account the length and severity of the inflammation around the implant is especially relevant in case of muscu­ loskeletal conditions such as a torn tendon or a cartilage lesion. In these diseases, the tissue requiring the repair is typically of poor quality, characterized by degeneration, reduced cell viability, preexisting inflammation, and abnormal ECM composition. Thus, the implantation of a foreign material should be carefully evaluated, to assess if any adverse response occurs. [10]. When a material is implanted, blood comes to contact with its surface, promoting provisional matrix formation and the following consequent events: acute inflammation, chronic inflammation, and granuloma and fibrous capsule formation. Depending on the size of the implant and its degradation products, large particles over 10 μm will cause foreign-body giant cell formation, while particles smaller than 5 μm are phagocytosed by macrophages. In the presence of a giant cell, macrophages will release various mediators of degradation, such as proteases, acids, and reactive oxygen intermediates. Cells in the surrounding tissue are likely to be affected by the acidic environment around the implant as well as the degradation enzymes specific to the foreign body [68]. Thus, while the size, chemistry, and degradation pattern of the material are important factors likely to influence the extent and nature of the inflammatory response, the pace and length of the degradation process may have an even greater effect. Currently available commercial silk sutures are considered nonabsorbable because they do not lose their tensile strength within 60 days of implantation, which is the time frame set for the classification of sutures [4]. However, it is well established that in vivo silk gradually degrades by the action of proteolytic enzymes associated with foreign-body response, and is completely absorbed within 2 years of implantation [4, 16, 95–98]. Nevertheless, one study has observed no disappearance of mass over 360 days of implantation [11], and a summary of case studies has reported the presence of silk suture fragments 7 years after surgery [67]. The pace and pattern of silk degradation is thought to be dependent on the processing, structure, size, and morphology of the scaffold [98]. The length of the process as well as its products is linked to the progression of the foreign-body response. The in vitro enzymatic degradation of B. mori silk fibroin films and fibers has resulted in a complex mixture of peptides with a wide range of molecular weights [95]. Regenerated fibroin scaffolds, and especially those with a lower fibroin concentration, have degraded more readily than native degummed fibers [98, 99]. Only a handful of studies specifically evaluated the inflammatory response to silk and its degradation products [18, 83, 100, 101]. In vitro assays have shown little or no macrophage stimulation by either regenerated [83, 100, 101] or native silkworm silks [101]. These studies have demonstrated a high compatibility of degummed (sericin free) silk, and pointed out sericin as the main incompatible element in B. mori silk. However, a recent study has shown sericin to be nontoxic and noninflammatory both in vitro and in vivo, eliciting a lower inflammatory response than a saline water control [102]. The exact role of sericin in the immune response to silk thus remains unclear. The published studies where silk was implanted in various animal models have also produced an ambiguous bank of data. The majority of these studies used a rabbit model, but, occasionally, rat and sheep models have been used [11, 12, 16, 25, 41, 48, 53, 54, 98, 103, 104]. Most of these studies have focused on the ability of the scaffold to support tissue regeneration and its mechanical robustness compared to a control. Predominantly, these studies have been short term (up to 20 weeks, see Figure 4) and have described excellent cell infiltration, good matrix synthesis, and organized neo-tissue formation around the implant. While many of these studies have not commented in detail on the inflammatory response, the reported tissue reactions varied greatly between those that did. A number of studies described the extent of inflammation and tissue reaction to both degummed and regenerated silkworm silk scaffolds as moderate, with some infiltration of macrophages, but with good interface between the regenerated tissue and the scaffold [11, 12, 25, 98, 103]. Others reported the formation of a thin, fibrous capsule around the implant [11, 53]. One study showed that scaffolds cast from lower concentrations of regenerated fibroin and processed in water rather then an organic solvent were better tolerated in terms of short- and long-term immune response [98]. A few studies pointed out a more severe inflammatory reaction upon the implantation of native, degummed silk fibers [16, 104]. Spelzini et al. [104] compared woven degummed B. mori silk to polypropylene meshes implanted in rat abdominal wall, and reported a stronger foreign-body reaction and a fast onset of profound fibrosis around silk over 90 days. This study has concluded that the level of immune response

Evaluation of Silk as a Scaffold for Musculoskeletal Regeneration – the Path from the Laboratory to Clinical Trials

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Figure 4 A pie chart of the length of the in vivo studies of silk scaffolds reviewed in this article [12, 25, 30, 41, 52–54, 98, 99, 103, 104]

remains a barrier for clinical use [104]. In another study, a vascular B. mori silk stent is described as having excellent patency 1 year after implantation in rats with good cell infiltration, but also showing substantial infiltration of macrophages and phagocytic phenomena around the remnants of the graft [88]. Liu et al. [53] reported strong inflammatory reaction to nondegummed silk fibers, which was much reduced upon coating fibers with gelatin. The author commented that tissue reaction to silk scaffolds has been “as good as catgut sutures” [53]. It is worth noting that compared to other sutures, catgut sutures are considered to elicit the highest inflammatory response, and are rarely used in modern clinical practice [61, 105–107]. These differing descriptions of inflammatory responses to silk scaffolds remain unexplained. It seems likely that differences are the result of study design, scaffold preparation, the choice of animal model, and tissue type. Most critical appears to be the difference between the immune response to regenerated and native silk, with the later being more frequently implicated with severe tissue reactions. However, more detailed reports of fibrosis, macrophage numbers and infiltration, as well as a comparison to existing implants and between tissue types may help to explain these gaps. Moreover, none of these studies showed the histology of neighboring tissues. Neither did they continue the period of study to include complete degradation of the scaffold. This is understandable because such studies are expensive and the results difficult to interpret. However, it is an important requirement to ensure that potential migration of degradation products does not occur or result in a remote body-site accumulation [10]. Finally, an important aspect of tissue reaction is amyloidosis. Amyloidosis is the deposition of abnormally folded proteins in tissues or organs, with a possible effect on normal tissue function as well as stiffening of the tissue. Amyloidosis is often a secondary complication of chronic inflammatory diseases such as arthritis and other musculoskeletal conditions [108–110]. Past studies compared amyloid fibrils and silk fibers [111–113], pointing out similarities in β-sheet content, folding, and aggregation. Two studies demonstrated the ability of regenerated silk from B. mori to enhance amyloidosis in mice [33, 114]. As these studies used liquid fibroin in combination with silver nitrate to induce amyloid aggregation, any direct comparisons to scaffolds from regenerated silk should be viewed cautiously. Nevertheless, in the light of these findings and the in vivo studies reviewed above, better characterization of the tissues implanted with silk scaffolds as well as tissues more distant from the implant is an essential part of assessing the safety of silk.

5.27.5 Summary As shown in the first part of this review, silk scaffolds, mostly from B. mori silks, have been established as excellent supporters of musculoskeletal regeneration, both in vitro and in vivo. Their excellent mechanical properties, combined with good support for cell growth, have been shown in numerous studies, providing a strong proof of concept for their future use as implants to repair bone, cartilage, ligaments, and tendons. However, to move silk onward to clinical trials, experimentation must move from proof-of­ concept studies to long-term preclinical evaluation of silk and silk-based materials not only as functional but also as safe implants. Moreover, the preclinical evaluation of silk scaffolds must address the major issues arising from past literature. Most importantly, the inflammatory reaction to silks must be better understood. Many papers rely on past assumptions that sericin is the immunogenic or incompatible element in silk [95, 99]. There is still not enough conclusive evidence to back that theory, with at least one recent study disproving the immunogenicity of soluble sericin [102]. A careful reconsideration of the exact tissue reaction to the different silk scaffolds is called for. In addition, experience from other implants has shown that slower-degrading polymers are more likely to elicit processes such as carcinogenesis and chronic inflammation [10], and that severe tissue reaction is tightly linked to the rate of degradation. In the case of silk, faster-degrading scaffolds from regenerated silk were better tolerated then slower degrading scaffolds of similar composition

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Table 1

Comparison of silk suture to regenerated and native silk scaffolds Commercial silk suture

Native, degummed silk fibers

Regenerated silk fibroin scaffold

Strongest point

Excellent handling characteristics

Excellent mechanical properties

Weakest point

Poor tissue reaction

Complete absorption

Normally thought to degrade within 24 months, occasionally detectable over longer term Unknown

Relative poor characterization, mixed results in vivo Contradicting reports, range of months/years

Easily modified and controlled. Can form sponges, gels, films Harsh reagents used during processing tensile properties reduced compared to native silk Could be adjusted to a few months, depends on porosity and fibroin concentration

Degradation products and their safety In vitro Toxicity Amyloid formation Inflammation

Carcinogenesis Hemocompatibility Allergy Genotoxicity Effect on distant tissues

Unknown

One study implicated leachables in modifying cell behavior Unknown

Largely considered nontoxic

Compared to other sutures considered to elicit strong foreign– body response One report indicated that silk suture encouraged tumor formation Thrombogenic Many reports of allergic reactions, exact source unknown Unknown Unknown

Mixed results. A few in vivo studies showed strong foreign body response Unknown

Unknown

A large range of degradation products. Degradation by α-chymotrypsin produced nontoxic degradation products No evidence of toxic effect in vitro Fibroin solution in combination with silver nitrate induced amylogenesis Reported moderate tissue reaction

Unknown

Thrombogenic Unknown

Inert to human plasma Unknown

Unknown Unknown

No Unknown

[98]. While native fibers were frequently reported to degrade extremely slowly and cause severe inflammation, regenerated silk showed a much faster degradation rate and a moderate immune response. These significant differences further highlight the need for a clearer separation between regenerated silk, native fibers, and silk sutures. A summary of major differences between silk scaffolds and sutures, as reviewed throughout this paper, is shown in Table 1. Better distinction between novel fibroin-based scaffolds and native silks would also help to distinguish them from traditional B. mori silk sutures, which have a complicated history of allergic tissue reaction and foreign-body response. This review also highlighted the need for more in vivo evidence of efficacy and long-term safety. There is still a need to characterize tissue response through the complete life of silk implants, in the relevant tissue as well as more distant tissues. As stated earlier, especially in damaged tissues such as torn tendons or cartilage lesions, the tissue requiring the repair is typically of poor quality, and testing under more realistic disease conditions may be vital for proving efficacy and predicting adverse events. This may require inducing disease in animal models as a better representation of the relevant condition [8]. Finally, the move from proof of concept to preclinical studies will also require further characterization of the complete content of native fibers. This will assist in revealing any sources of incompatibility as well as understanding the composition, fate, and safety of the degradation products of silk. Moreover, a clearer view of silk degradation products and their safety may help in the future to design a new generation of safe, predictable, novel scaffolds.

Acknowledgments The authors would like to thank the NHIR Biomedical research unit for funding Prof. Andrew Carr and Dr. Osnat Hakimi, as well as the European Research Council (grant SP2-GA-2008-233409) for funding Prof. Fritz Vollrath.

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Relevant Websites http://www.fda.gov – FDA: U.S. Food and Drug Administration; General regulation of medical devices. http://emedicine.medscape.com – eMedicine; Basic information about sutures.