Will silk fibroin nanofiber scaffolds ever hold a useful place in Translational Regenerative Medicine?

Abstract

Presently, some view silk fibroin-based biomaterials as obsolete, being outperformed by a host of newly discovered biomaterials. But several lines of evidence support the notion that silk fibroin proteins, especially those from B. mori and spiders and their recombinant forms, particularly in the form of electrospun nanofiber scaffolds, still represent promising tools for human tissue engineering/regeneration. Inevitably, the allure of recently reported biomaterials turns away many scientists and resources from the aim of more deeply elucidating the biological interactions of the various kinds of silk fibroin nanofiber scaffolds in vivo. But, even the biological features of newly reported biomaterials are not investigated in adequate depth. Hence, collaborative efforts among biomaterialists, biomedical experts, and private firms must be undertaken on a much greater scale than hitherto done to assess the real usefulness of silk fibroin proteins, thereby allowing or denying their useful introduction into the fields of Translational Regenerative Medicine.

 

The ideal gold standard for purposes of human tissue engineering/regeneration are deemed to be biomaterial nanofiber scaffolds endowed with a set of optimized features in connection with surface properties, biocompatibility, gas permeability, biodegradability, mechanical strength, immunogenicity, foreign body response (FBR), and release of growth factor(s) and/or drug(s). Amid the biomaterials candidating for such nanofiber scaffolds, the silk fibroin proteins (SFs), have been objects of a growing interest [1,2], as demonstrated by the trend of the number of publications appeared in the scientific literature during the last ten years (Figure 1). SFs are natural proteins produced by wild and domesticated silkworms, spiders, honeybees, wasps, and ants [3-7]. Variants of SFs have been genetically engineered to produce novel biomaterials [8]. They share repetitive amino acid sequences of the poly(Ala) or poly (Gly-Ala) type arranged in antiparallel beta-sheet structures [9], are biodegradable in vitro and in vivo [10-13], very little inflammogenic [14,15] and can be electrospun into pure SF nanofibers [16-22] or blended with other biomaterials, e.g. poly(L-lactic acid), poly(ethylene oxide), poly(ε-caprolactone), hydroxybutyl chitosan, heparin, collagen, gelatin, etc. [23-37].

Figure 1.

Trend of publications on SF nanofiber scaffolds for biomedical applications

The electrospun SF scaffolds have been reported to exhibit good cell biocompatibility [38] and their structure and physicochemical features were shown be deeply influenced by the treatments SF undergoes prior to and/or during electrospinning [4,21,25,39-41]. Application of SF nanofiber scaffolds has been suggested for the engineering and regeneration of both soft tissues, like vascular grafts, nerves, skin wounds [42-54], and hard tissues, like tendons, ligaments, bone and cartilage [16,55-65], although in the latter instances SF was also used as microfiber or sponge and mixed with other biomaterials. In these tissue regeneration attempts bone marrow mesenchymal stem cells have often been seeded onto the scaffolds to observe their differentiation into the several types of the connective tissue or into Schwann peripheral nerve cells [49]. To enhance cell adhesion, spreading, and proliferation, SF nanofiber scaffolds were also functionalized with various bioactive peptides (RGD, BMP, etc.) [16,54,66].

Notably, most if not all of the authors claim to have been successful in their endeavors. So, why the SF nanofiber scaffolds have not as yet found their way to the application in the clinical settings?

What is missing?

It should not be overlooked that most of the published studies on the topic mainly concern the still ongoing efforts to improve the technical procedures used to prepare the scaffolds and hence the scaffolds themselves and to assess their physicochemical properties. Conversely, the interactions of the scaffolds with living cells in vitro or in vivo, especially the long-term ones, often if not mostly appear as shallow appendages of the biomaterialistic studies. Is this an indication that we are still in too an early phase to have set up appropriate scaffolds? This scientific “immaturity” is moreover compounded by the fact that the biological tests have been preferentially carried out on rodent cells of rats or mice, indeed imperfect mirrors of the corresponding human tissues. In such models, the local and general responses to the implanted SF nanofiber scaffolds, as well as the timing and rates of the degradation of the latter have been poorly assessed. On the whole, while the technically complex questions related to the characteristics and production of the nanofiber scaffolds keep being targeted, the as well or even more intricate biological interactions of the same scaffolds with living cells, tissues, and organisms have by and large remained in the shades. This has somewhat delayed or hampered the onset of an applicative era for the SF nanofiber scaffolds. As a consequence, in some people, this has generated the idea that SFs are old-fashioned biomaterial relics, interesting maybe but not so functional: hence, they should be superseded by novel, more fashionable, and more readily applicable biomaterials.

What the remedy might be?

In the opinion of the authors, this negative outlook is hasty and not founded enough. First of all, it should be recalled here that many human proteins expressed by both epithelial and connective tissue cells exhibit biologically significant (i.e., with very low E-Values and P values) homology sequences with heavy-chain B. mori SFs (just as an example, see the 49 human proteins listed in Table 1). Such proteomic data underlie the reason why surgical stitches made of degummed (sericin-deprived) SF are not immunogenic in humans [67]. In addition, SF-based scaffolds have been shown to favor angiogenesis, an feature essential for tissue repair/regeneration [15,68]. Hence, these important pieces of evidence are not to be overlooked in the perspective of the clinical application of SF-based scaffolds. Moreover, a huge hoard of promising data concerning SF-based biomaterials has been accumulating during recent years. To make the jump to the applicative settings some more technological improvement leading to more robust and reliable production techniques, a deeper knowledge of structure/function relationship of SF nanofiber scaffolds and, as mentioned above, an increase in the understanding of the biological interactions are all is needed. Therefore, to seriously overcome such hurdles and reach a definitive assessment of the opportunities (or, unluckily, lack of them) of the SF nanofiber scaffolds in human tissue repair/regeneration, it should be realized that biomaterialists and cell/tissue/animal/human biology experts must solidly cooperate with each other while absolutely avoiding the hard to resist temptation to assume each other’s role, as the skills required for either job are highly complex and specific. By each one sticking to his/her respective role, it would be easier to carry out compound and articulated research projects aimed at scalar, chondrogenesis, osteogenesis, etc.). These major research projects would entail several successive rounds of structural and fly optimizing and standardizing the SF nanofiber scaffolds according to the targets they are aimed at (wound healingunctional improvement of the SF nanofiber scaffolds based upon the results gained by assessing both in vitro and in vivo the biological properties of their previous versions. Thus, through the proper use of the specific expertise of either group, the time required to attain optimized (or nearly so) scaffolds should be significantly shortened. This recursive testing and optimization of the scaffolds would finally open the way to preclinical testing of the optimized scaffolds in large mammals (e.g. dog, sheep, etc.). The assessment of the local tissue engineering/regeneration and local inflammatory and FBR responses and of general immunological (if any) reactions to the grafted scaffolds in such mammals would allow their definitive optimization. At this step, phase I clinical trials on selected cohorts of human patients would be feasible and fully justified, and their results would (or would not) lead further to phase II and III clinical trials and, eventually, to the approval of the scaffolds in their final formulations for human use by the FDA. It is implicit that to implement such a scheme large monetary resources would be needed and the collaboration between international research groups more than welcome would be mandatory.

Table 1.

A partial list of human proteins endowed with biologically significant sequence homologies to heavy-chain SF from B. mori

Conclusions

On the basis of the available lines of evidence, SF proteins, especially the most studied ones from B. mori and spiders, and their genetically recombinant forms can still be considered as promising tools and not outdated biomaterials, for human tissue engineering/regeneration. But it is clear that efforts on a much greater scale than up till now are needed to definitively assess the effective usefulness of such tools, thereby allowing (or negating) their successful introduction into the fields of Translational Regenerative Medicine (i.e. from the lab bench to the patient’s bed). A factor complicating the picture is that the SF nanofiber scaffolds may contain additional biomaterials and be molded into manifold morphologies according to their specific aims. No doubt, these efforts require the wide cooperation of international groups with deep specific expertise in their respective fields, i.e. biomaterial technologies and animal and human cell and tissue biopathology, respectively. To such projects private firms should also partake given the huge economic interests involved if the translation of SF-based nanofiber scaffolds to the clinical settings is made possible.

Unavoidably, the incessant discovery of novel biomaterials other than the SFs, while advancing our knowledge in basic sciences, takes away scientists and resources from more deeply focusing on the potential applications of the SF nanofiber scaffolds. But the biological features of these newly discovered biomaterials are not generally studied in greater depth either. Hence, from the standpoint of Translational Regenerative Medicine, it remains undecided whether eventually they might or not be superior to the SF nanofibers. Therefore, although the currently available choices are manifold, it is conceivable that a considerable loss for Human (and Veterinary Medicine) could stem from not fully investigating the real clinical opportunities well devised and tested SF nanofiber scaffolds would be likely to offer.