We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.

The relative paucity of techniques currently available to repair bone tissue necessitates the development of innovative and more effective clinical strategies. [1,2] Of these, the combined integration of macroporous scaffolds, primed human-cell populations, and growth factors to organize and promote tissue formation is a particularly attractive approach. [3,4] However, bone tissue engineering is currently compromised by its inability to produce load-bearing scaffolds. For example, collagen scaffolds are used for a range of osteogenic applications, [5][6][7] but exhibit a compressive strength of ca. 0.034 MPa, [8] approximately three orders of magnitude lower than that of cancellous bone with values between 10 and 50 MPa. On the other hand, pure calcium phosphate mineral-based scaffolds, which currently dominate the commercial bone substitute materials market, lack a fibrillar protein component and are correspondingly brittle, typically failing catastrophically at compressive loads of less than 5 MPa.Recent studies have explored the possibility of replacing collagen with silk-based resorbable implants. [9][10][11][12] Silk is a fibrillar protein with excellent biocompatibility and mechanical strength, [13] and methodologies exist to convert silk fibers into regenerated silk fibroin solutions that can be subsequently reconstituted into macroporous 3D architectures with b-sheet secondary structure by salt leaching, gas foaming, extrusion layering or freeze drying. [9,[13][14][15][16][17][18] Although these scaffolds are potentially suitable for tissue engineering, similarly to collagenbased biomaterials the reconstituted silks are compromised by low mechanical strength, due in part to partial degradation of the native protein structure during fibroin dissolution. Moreover, attempts to impart significant mechanical reinforcement by calcium phosphate mineralization have not been very successful, due to poor adhesion and integration at the protein/mineral interface. [19,20] As a consequence, more advanced uses of silk are currently focused on novel delivery devices for morphogens, cytokines, and cell populations in models of bone defects. [21][22][23][24][25] In contrast, we present herein the first example of a silk/calcium phosphate macroporous scaffold that is load-bearing with mechanical properties comparable to cancellous bone. The mechanical strength is far in excess of other materials previously produced, and is achieved through the use of high-quality silk fibroins [26] and an integrated procedure for gelation, freezing, and mineralization. Moreover, we demonstrate the effectiveness of these load-bearing materials as nonpyrogenic osteoregenerative scaffolds by in vitro and in vivo testing, and suggest that such materials represent a new class of potentially implantable alternatives to the use of allograft and autograft procedures, for example in surgical applications, where immediate load bearing is required.Silk/calcium phosphate macroporous scaffolds were prepared by freezing phosphate-containing aqueous gels...

Lipoteichoic acid (LTA) is a major immunostimulatory molecule in the cell wall of Gram-positive bacteria. Adhesion of LTA to a polystyrene surface drastically increased its immunostimulatory potency in human whole blood in comparison to soluble LTA, although only 1% of the LTA had bound, as determined using rhodamine-labelled LTA. The release of the proinflammatory cytokines IL-1beta, TNF and IL-6 and the chemokines IL-8 and G-CSF was increased 2- to 10-fold, but IL-10 release was unaltered. This presentation effect was not shared by lipopolysaccharide (LPS) or other toll-like receptor 2 agonists and was less pronounced in polypropylene vessels. LTA did not induce cytokine release in silicone-coated borosilicate vessels, but covalent coupling of LTA to polystyrene beads restored cytokine induction in these vessels, indicating that presentation of LTA on a surface is in fact essential for its immunostimulatory potency. This novel aspect of presentation as a factor in the recognition of LTA may reflect the physiological situation in the bacterial cell wall, where LTA is anchored in the bacterial membrane and projects through the peptidoglycan. In practical terms, contamination of medical devices with components of Gram-positive bacteria may pose an underestimated inflammatory risk.