Target Tissue Engineering Applications

This section will focus on tissue engineering and regenerative applications with the diverse set of silk-based biomaterial formats. Cases, illustrating recent advances toward the regeneration of commonly targeted organs will be discussed below.

2.2.2.1 Bone

Bones are mineralized, highly organized tissues with essential functions in support, motility, protec-tion, hematopoiesis and calcium homeostasis. Structurally, compact (cortical) bones have supportive functions, while spongy (cancellous) bones fulfill metabolic functions (Sandy et al., 2002). Bone engi-neering poses challenges because of the morphological, structural, and functional complexity. Bone repair however is a frequent procedure in the medical world. Tissue engineering offers alternatives that could avoid repeated surgery and reduce second site morbidity.

Processing techniques currently used to yield silk based biomaterials allow for the generation of scaf-folds that mechanically match or compare to those of the native tissue. Porous 3D silk fibroin scafscaf-folds emerged as optimal candidates for bone regeneration (Kim et al., 2005a, Meinel et al., 2004, 2005). These scaffolds are commonly obtained by either all aqueous or HFIP processing followed by salt leaching, gas foaming, and freeze-drying, to generate the porous structures (Kim et al., 2005a). Depending on the processing path, different porosities could be obtained which translate into different mechanical properties and degradation rates. Typically aqueous processing yields rougher scaffolds, with intercon-nected pores and higher mechanical parameters (Kim et al., 2005b). Nevertheless, both in vitro and in vivo, these aqueous-based scaffolds degrade at a faster rate than their HFIP processed counterparts (Kim et al., 2005b, Wang et al., 2008). This correlates with the extent of beta-sheet content formed dur-ing processdur-ing, which is a major determinant of different degradation rates (Kim et al., 2005b, Nazarov et al., 2004).

The relationship between scaffold degradability and human mesenchymal stem cell (hMSC) osteo-genesis in in vitro dynamic cultures has been investigated (Park et al., 2010). Scaffolds with different degradation rates were obtained by the aforementioned processing methods. Scanning electron micros-copy, von Kossa, type I collagen staining and calcium content determination showed extensively miner-alized extracellular matrices (ECM) formed in the scaffolds designed to degrade more rapidly. Levels of ECM osteogenic markers were also significantly higher in the more rapidly degrading scaffolds than in the more slowly degrading scaffolds over 56 days of study in vitro. Metabolic glucose and lactate levels were also scaffold-dependent, with the more rapidly degrading scaffolds supporting higher levels of glucose consumption and lactate synthesis by the differentiated cells, in comparison to the more slowly degrading scaffolds.

Dynamic culturing conditions of hMSCs were also used to engineer bone implants for critical sized calvarial bone repair in nude mice (Meinel et al., 2004, 2005). The engineered bone implants displayed trabecular-like bone networks with ECM similar to the native tissue. Good integration was observed after 5-weeks postimplantation, and constructs stained positive for osteogenic markers (sialoprotein,

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osteocalcin, and osteopontin). The controls (scaffolds alone, unfilled defects) did not support regenera-tion or were less substantial in terms of bone formaregenera-tion, as was the case for freshly cell-seeded scaffolds.

In a critical size femoral defect study organic solvent-based silk scaffolds infused with bone mor-phogenetic protein (BMP-2) and seeded with hMSCs, with or without prior osteogenic differentiation, induced significant bone morphogenesis compared to the untreated controls (Kirker-Head et al., 2007).

BMP-2 addition induced more bone formation than observed in the noninfused scaffolds. Moderately good bridging between the native and regenerated bone was attained.

These few examples are illustrative of the advances made toward recreating functional bone and also pinpoint the issues that still need to be address. Scaffold preparation methods, scaffold mechanical, physico-chemical and biological properties, cell culture conditions, and the addition of growth factors are all parameters that interplay in the tissue engineering process of bones in order to recapitulate the properties of native tissues.

2.2.2.2 Cartilage

Cartilage is a stiff, inelastic tissue comprised of chondrocytes that secrete abundant collagen type II rich matrices. This system is avascular, with nutrient influx and efflux dictated by diffusion and facilitated by tissue mechanics (compression, elongation). The lack of an abundant nutrient supply translates into a system with low and slow regenerative capacity. Cartilage damage caused by developmental abnormali-ties or immunological disorders, trauma or aging, is associated with chronic pain and gradual loss of mobility. Current treatment options are aimed at reducing pain and decreasing cartilage degradation rates, but they fail to restore normal cartilage function.

Tissue engineering approaches using both stem and primary cells have been employed on this direc-tion. Porous, aqueous-based silk scaffolds seeded with hMSCs and primary chondrocytes were studied in vitro for cartilage regeneration (Wang et al., 2005, 2006b). Stem cells cultured on silk scaffolds under static conditions underwent chondrogenesis as evaluated by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-cartilage-specific ECM components (Wang et al., 2005). Upon 3 weeks in culture, most cells acquired a spherical mor-phology (essential for the synthesis of cartilage-specific ECM components), and were embedded in scaffold-specific lacunae-like niches resembling the native tissue architecture. Moreover, collagen type II distribution in the hMSC-silk scaffold constructs resembled those in native articular cartilage tissue.

No calcium deposition was detected by von Kossa staining indicating the lack of osteogenesis. However, dexamethasone and transforming growth factor (TGF)-beta3 were supplemented in the culture media of these constructs, while they were absent in the controls. This fact encumbers the interpretation of the data and the separation of scaffold-cell versus additive effects on chondrogenesis.

Aqueous-based porous silk fibroin scaffolds were also studied when seeded with adult human chon-drocytes (hCHs) (Wang et al., 2006b). The attachment, proliferation and re-differentiation of hCHs in the scaffolds in serum-free chemically defined medium with TGF-beta1 was evaluated based on cell morphology, levels of cartilage-related gene transcripts, and the presence of cartilage-specific ECM.

Compared to hMSC attachment, primary cells attached more slowly and cell density appeared critical for the re-differentiation of culture-expanded hCHs in the silk fibroin scaffolds. Moreover, there was an upregulation in the level of cartilage-related transcripts (aggrecan core protein, collagen type II, transcription factor Sox 9 and collagen type II/collagen type I ratio) and deposition of cartilage-specific ECM in constructs initiated with higher seeding density compared to their hMSC seeded counterparts.

In contrast to hMSCs, all hCH cells adopted spherical morphologies after a 3-week culture period. This study indicates that primary chondrocytes might be competitive for tissue regenerative applications, but these results will need to be further confirmed in in vivo models.

2.2.2.3 Tendon/Ligament

Tendons and ligaments are both fibrous tissues comprised of fibroblasts arranged in parallel that secrete a collagen type I and proteoglycan-rich matrix. Tendons connect muscles to bones, ligaments link bones

to bones and both tissues need to sustain significant mechanical stress. This is achieved by a strict hier-archical organization starting with collagen fibers assembled into microfibrils, microfibrils into subfi-brils, subfibrils into fibrils interspersed with fibroblasts to form fascicles. Fascicles are then clustered together to form the tendon or ligaments.

Tendon and ligament repair is especially prevalent in sports medicine. Challenges arise in the ability to recreate a mechanically functional tissue. Classical treatment options imply lengthy recovery time, arthritis, donor site morbidity, and degenerative joint disease. Lack of mechanical stimulation after recon-structive procedures frequently leads to undesired inter-hierarchical structural adhesions that result in impairment or loss of function. Tissue engineering approaches in this area are still in their infancy, due to the complexity of the tissue. To date, attempts were made to recapitulate the native tissue architecture by employing fiber-like biomaterials. An initial effort in this direction used a wire-rope model designed silk-fiber matrix to engineer anterior cruciate ligaments (ACL)-like structures (Altman et al., 2002). The matrix matched the mechanical requirements of native human ACL including the fatigue performance.

In addition, scanning electron microscopy, DNA quantitation and detection of collagen types I and III and tenascin-C marker expression indicated hMSC attachment, expansion and differentiation. A dif-ferent approach used RGD-modified silk sutures cultured with human tenocytes (Kardestuncer et al., 2006). These substrates supported increased cell adhesion after 3 days when compared with unmodified silk fibers and tissue cultured plastic. Collagen type I and decorin transcript levels were also higher on the RGD-modified sutures compared with unmodified silk and tissue culture plastic at 6 weeks. These studies indicate the compatibility of silks for tendon and ligament regeneration and repair.

2.2.2.4 Skin/Wound Healing

Since skin covers the entire surface of the body, it is highly prone to injury. Skin regeneration can be less challenging compared to other organs and tissues, with an architecture that is fairly uncomplicated, and achievable mechanical properties, such as an elastic modulus of ~120 kPa (Diridollou et al., 2000). A myriad of synthetic and natural biomaterials have been employed as wound dressings and regenerative scaffolds, each with its merits and pitfalls. Silk-based scaffolds have also been employed for this purpose in various forms, including epidermal growth factor (EGF)-releasing silk mats (Schneider et al., 2009).

A human skin-equivalent wound model, displaying similar architecture and molecular and cellular healing mechanisms as the native organ, was used to evaluate the silk-based constructs. Silk mats main-tained their structure and biocompatibility, slowly released EGF and increased wound closure time by 90% compared to no treatment. These results establish silks as potential candidates for skin regenera-tion and wound repair and open avenues for testing novel silk-based biomaterial formularegenera-tions.

2.2.2.5 Cornea

The cornea—the transparent structure covering the anterior part of the eye—is responsible for approxi-mately two-thirds of the eye’s total optical power. Corneal blindness represents a major issue world-wide. Therapeutic approaches utilize corneal grafting, commonly from cadaveric donors. However, by 4–5 years post implantation the immunological rejection rate is ~25% and continues to increase over the life of the patient (George and Larkin, 2004, Nishida et al., 2004). Synthetic keratoprostheses, con-structed of poly-2-hydroxyethylmethacrylate, are currently available yet they also exhibit a relatively high host rejection rate (Ilhan-Sarac and Akpek, 2005, Myung et al., 2007). Corneal tissue engineering builds on the native tissue architecture. The corneal transparency is a reflection of its stromal organiza-tion, with an extracellular matrix consisting of hybrid type I/V collagen fibrils, extraordinarily uniform in diameter and regularly arranged into a pseudolattice. Moreover, the fibrils are kept at defined dis-tances by proteoglycans (Knupp et al., 2009).

Silk film biomaterials were used to recreate the stacked architecture of the cornea (Lawrence et al., 2009). Films were 2 µm thick mimicking corneal collagen lamellae dimensions and porous to permit nutrient trans-lamellar diffusion and promote cell−cell interactions. In addition, film surfaces were pat-terned to guide human and rabbit corneal fibroblast cell alignment. The final constructs sustained cell

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proliferation, alignment and corneal extracellular matrix expression, were optically clear and had good mechanical integrity indicating the suitability of silks for such applications.

2.2.2.6 Peripheral Nerves

The peripheral nervous system consists of cord-like structures containing bundles of nerve fibers that carry information from limbs and organs to the spinal cord and back. In contrast to the brain and spinal cord that have very limited healing capacity, peripheral nerves can regenerate, even when completely severed, resulting in complete or nearly complete recovery of the patient. However, in some cases, the process is slow enough to cause the affected organ to be paralyzed or to atrophy. Autologous grafts are typically used in peripheral nerve reconstructive surgery (Subramanian et al., 2009). However, auto-grafts have drawbacks such as limited availability, mismatch of donor-site nerve size with the recipient site, neuroma formation and lack of functional recovery. As an alternative, allogenic grafts from cadav-ers address the availability issue, but often cause immune rejection.

A silk fibroin conduit loaded with neurotrophic factors was recently evaluated in a small nerve gap repair model (Madduri et al., 2010). This system was functionalized with aligned and nonaligned silk fibers to aid axon orientation. Both sensory and spinal cord motor neurons from chick embryos exhib-ited increased length and rate of axonal outgrowth parallel to the aligned nanofibers. Glial cells from dorsal root ganglions proliferated and migrated in close association and even slightly ahead of the outgrowing axon while on nonaligned fibers both axonal and glial growth was slower and randomly oriented. These data suggest that silk fibroin-based conduits have the potential to enhance functional recovery of injured peripheral nerves and may offer a viable treatment option for rapid recovery.