Silk fibroin and ceramic scaffolds: Comparative in vitro studies for bone regeneration

Abstract Synthetic bone void fillers based on calcium ceramics are used to fill cavities in the bone and promote bone regeneration. More recently, silk fibroin (SF), a protein polymer obtained from Bombyx mori silkworm, has emerged as a promising material in bone void filling. In this work, we have compared the safety and efficacy of two types of silk fibroin‐based bone void fillers with currently used and commercially available ceramic bone void fillers (based on calcium sulphate, beta tricalcium phosphate, and beta tricalcium phosphate with hydroxyapatite). Further, we have also evaluated these two types of SF scaffolds, which have strikingly different structural attributes. The biocompatibility of these scaffolds was comparable as assessed by cytotoxicity assay, cellular adhesion assay, and immunogenic assay. Ability of the scaffolds to support differentiation of human mesenchymal stem cells (hMSCs) into an osteoblastic lineage was also evaluated in an in vitro differentiation experiment using reverse transcriptase polymerase chain reaction analysis. These results revealed that cells cultured on SF scaffolds exhibit higher expression of early to late markers such as Runx2, BMPs, collagen, osterix, osteopontin, and osteocalcin as compared with ceramic‐based scaffolds. This observation was further validated by studying the expression of alkaline phosphatase and calcium deposition. We also show that scaffolds made from same material of SF, but characterized by very different pore architectures, have diverse outcome in stem cell differentiation.

may also be caused due to a tumor or an infection in the bone. To accelerate the healing of bone in these clinical conditions, the cavities are typically filled with a bone void filler. A variety of natural and synthetic materials have been used as bone void fillers. More than 50% of the materials used for bone void filling are based on calcium ceramics. These include materials such as hydroxyapatite (HA), calcium sulphate (CaSO 4 ), beta tricalcium phosphate (β-TCP), and their composites/blends. [2][3][4][5][6][7] HA has been extensively used for bone void filling applications for several decades. It is biocompatible and bioinert, that is, it does not induce an inflammatory response from the host tissue and is generally well tolerated at the implantation site. It has also been shown to support new bone formation. However, HA has an extremely slow rate of bioresorption and remains at the implantation site for several years. This has been a cause for concern and more recently other alternatives are being evaluated for this application. β-TCPand CaSO 4 -based bone void fillers overcome this problem of slow resorption of HA. These materials have been demonstrated to also exhibit excellent support for the bone regeneration. However, both β-TCP and CaSO 4 have extremely fast rates of bioresorption and are found to resorb within a few months after implantation. 7,8 This faster rate of resorption results in other complications such as incomplete filling of defects, poor quality of new bone formation, and sometimes also secondary fractures. 6,9,10 Thus, there is an active interest to develop novel bone void filling materials that overcome these limitations of existing materials.
Silk fibroin (SF), a natural polymer extracted from the Bombyx mori silkworm cocoon, has been explored as a promising material for bone void filling applications. 11 SF has exceptional thermomechanical properties, inherent and proven biocompatibility, easy processability, and controlled rate of bioresorption. Several researchers have demonstrated innovative processing protocols to make scaffolds of SF and have shown that these materials support new bone formation. [12][13][14] has also been blended with other biopolymers and bio-ceramics and these composites have also shown promising results in bone regeneration. 13 These scaffolds produced from SF and its blends/composites have varying porosities, pore architectures, and pore sizes. The scaffolds also exhibit a broad range of mechanical properties-for example, compression modulus varying from 0.1 MPa to >50 MPa. The conformation of the SF protein can also be controlled using various physical and chemical treatments and it has been shown to affect the mechanical and bioresorption characteristics of the scaffold. 15 In spite of the large volume of literature on SF and ceramics scaffolds for bone regeneration, there are not many studies that compare and contrast the ability of these scaffolds to support bone regeneration. Thus, the objective of this study is twofold. The first objective is to compare the ability of SF scaffolds vis-a-vis calcium-based ceramic scaffolds to support bone regeneration. This was done using in vitro assays that monitored the differentiation markers of new bone formation. The second objective is to compare the performance within two silk scaffolds, which have strikingly different structural attributes in bone regeneration. We selected three representative commercial ceramic bone void fillers that had a chemical composition consisting of CaSO 4, 16 β-TCP, 17 and a composite of β-TCP-HA. 18 These materials were selected since they are commercially available globally and are preferred products by several clinicians performing bone void filling surgeries. Further, we prepared two different types of SF scaffolds. An SF microparticle-based scaffold was prepared as per the protocol described in the study by Nisal

| RESULTS
Here, we compare the performance of the SF scaffolds vis-à-vis the conventionally used and commercially available calcium-based ceramic bone void fillers in bone regeneration using in vitro techniques. Further, we also used two types of SF scaffolds lyophilizedregenerated silk fibroin (L-RSF) and microparticle-regenerated silk fibroin (M-RSF), with significantly different mechanical and structural properties.
The bioceramic, L-RSF and M-RSF scaffolds vary in their pore size, porosity, and mechanical performance and crystallinity index.
These properties were measured using standard protocols described in detail in our earlier manuscript Nisal et al. and have been tabulated in Table 1. 19 The properties have also been discussed in Section 4 of the manuscript. Photographs of all scaffolds used in the study are included in Figure S1A. The bioceramic materials were used as is or as per the protocols described by the manufacturer. The material of construction of both silk scaffolds (L-RSF and M-RSF) is a natural protein polymer-SF. L-RSF scaffolds have significantly higher porosity as compared to M-RSF. L-RSF scaffold has random pores as shown in Figure S1B. Compression modulus of L-RSF scaffolds is significantly lower than M-RSF (Table 1).
The in vitro studies were conducted at two levels, assessment of biocompatibility and assessment of efficacy in supporting differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts.
2.1 | Biocompatibility testing 2.1.1 | Cytotoxicity testing In vitro cytotoxicity testing was done as described in ISO-10993 to evaluate the overall biocompatibility and safety of the biomaterials.
This was carried out using both direct contact and extract contact methods. Organo-Tin PU is a known cytotoxic material and hence used as a positive control and HDPE, known to be nontoxic was used as negative control for cytotoxicity, for direct contact method.
Extracts of these materials were also assessed for cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results of cytotoxicity testing using direct contact and extraction method are summarized in Figure 1(a,b), respectively. L929 cells in contact with both silk scaffolds (L-RSF and M-RSF) displayed comparable viability with plate control, all benchmarking products and negative control (HDPE) (Figure 1(a)). Cell viability for Organo-tin PU sample was found to be <25% and this confirmed the validity of the experiment. Extracts of both silk scaffolds (L-RSF and M-RSF) exhibited >90% viability (Figure 1(b)). These results indicate that both L-RSF and M-RSF scaffolds or their extracts are not cytotoxic. Also, the viability of cells in both studies is at par with the commercial products used for benchmarking. However, when the silk scaffolds were compared with the ceramicbased material, M-RSF showed comparable cellular adhesion with CaSO 4 and β-TCP-HA, but marginally lower cellular adhesion was seen when compared to β-TCP.

| Evaluation of inflammatory response :
Inflammation is the first physiological response upon implantation and hence it is necessary to assess the inflammatory response of any implantable material. An assay based on mouse macrophage cell line RAW 264.7 was used. A negative control in the form of tissue culture plate was incorporated in the experiment. Lipopolysaccharide (LPS) is known to induce inflammatory response and was therefore included as a positive control. All scaffolds were exposed to a monolayer of macrophage cells for defined time intervals, to allow cells to elicit immune response. Tumor necrosis factor alpha (TNF-α) and T A B L E 1 Comparative analysis of ceramic-based scaffolds (CaSO 4 , β-TCP, and β-TCP-HA) with silk-based scaffold (L-RSF and M-RSF) interleukin 1 beta (IL-1β) levels were quantified on days 2, 7, and 14 and the results are summarized in Figure 3.  (Table S1). These scaffolds also have vastly different microarchitectures ( Figure S1). These experiments allowed us to compare the performance of scaffolds based on the same material, which are differentiated by micro-architecture, porosity, and mechanical properties on osteoblast differentiation while benchmarking the performance with commercially available ceramic-based bone void fillers.

| Proliferation estimation by MTT assay
During the 28 days of experimental duration, hMSCs proliferate and also differentiate into osteoblasts via osteoblastogenesis. The process IL-1B expression TNF-alpha expression

| Gene expression studies of osteoblast differentiation markers
Scaffolds ability to support osteoblastogenesis was evaluated by mea-  Figure 5(a)) but did not support sustained proliferation and differentiation throughout the experiment ( Figure 5(b,c)). These results also corroborate with the ALP activity and Ca 2+ deposition data

| ALP expression and Ca 2+ deposition
Osteoblasts are the main cell type involved in new-bone formation.
Osteoblasts actively participate in matrix synthesis and bone minerali-    Figures 4 and 5).. [30][31][32][33][34][35] hMSCs cultured on all scaffolds supported proliferation. It must also be noted that the proliferation was found to be the lowest on the  29 The microarchitecture, including mechanical properties was distinct for all scaffolds (described in detail in Supporting Information). A time-dependent enhancement in the gene expression of the markers was seen in the cells cultured on all bone void fillers. As can be seen in the data summarized in Table 1, the expression of all early, early-to-mid, and early-tolate markers was found to be the lowest for β-TCP-HA scaffold. For most markers, the expression was found to be comparable for CaSO 4 and β-TCP scaffolds. The L-RSF scaffold showed expression levels higher than these ceramic scaffolds and the M-RSF scaffold outperformed all the scaffolds by at least 2× to 3× expression levels. Correspondingly, the ALP expression and calcium deposition or mineralization was also better supported by SF scaffolds as compared to other bone void fillers, with M-RSF showing significantly better performance as compared to L-RSF.
Mechano-transduction is a process that converts the mechanical stimuli from the scaffold stiffness into a chemical response. The stiffness of a scaffold is a key "passive" mechanical cue that affects stem cell differentiation. 29 Mechanical properties, pore size, and porosity were measured as described in the study by Nisal et al.. 19 Both scaffolds are described in detail below and their characteristics have been summarized in Table S1.
M-RSF scaffolds. M-RSF is a scaffold of fused microspheres (that are crystalline and solid) exhibiting~40% bulk porosity, interconnected pore structure and compressive modulus equivalent of cancellous bone (described in greater detail in Refs. 19,40,41). In brief, microparticles of SF were prepared by using a two-solvent system. The microparticles are monodispersed and have diameter of around 500-600 μm. These microparticles are highly crystalline (Crystallinity index = 1.6 ± 0.1) and nonporous. The scaffold was prepared by fusing these SF microparticles in a cylindrical mold using aqueous SF solution. The compression modulus of the scaffolds is 70 ± 6 MPa (dry) and 18 ± 2 MPa (wet) and have a bulk porosity of~40%-44%.

Ceramics-based bone void fillers
Various ceramic-based commercial bone void fillers were used as is or prepared as per protocols described by the manufacturer. We selected  Table S1.

| Assessment of in vitro inflammatory response
In vitro inflammatory response was assessed by using an assay based on RAW 264.7 cells (procured from NCCS, Pune, India). Cells were seeded at a density of 10,000 cells/well. These cells were

| Efficacy of scaffolds in supporting differentiation of hMSC's into osteoblasts (bone cells)
Effectiveness of the scaffolds in supporting proliferation and differentiation of hMSC's was evaluated. The following parameters were mon- Supernatant was transferred in 96-well plate and absorbance was measured at 405 nm. Assays were performed in triplicates and each assay was performed at least thrice to validate the observations. Data were expressed as mean ± SD.

| CONCLUSION
Synthetic bone void fillers are increasingly being used to fill cavities in the bone. In this work, we used a set of in vitro tests to assess the safety and efficacy SF-based bone void fillers vis-a-vis the currently used calcium ceramic-based bone void fillers. We selected two types of SF scaffolds that differed in their pore architecture, bulk porosity, and mechanical properties such as compression modulus. It can be concluded here that all scaffolds, irrespective of the chemical composition and physical characteristics, were noncytotoxic and supported cellular adhesion.
The scaffolds did not elicit any inflammatory response as monitored through the expression of cytokines such as TNF-α and IL-1 β. We also monitored the expression of several differentiation markers and/or transcription factors to evaluate the ability of the scaffolds to support bone tissue regeneration. All the scaffolds were found to support early stage of differentiation of hMSC's. The expression of differentiation markers was found to be lowest in the β-TCP-HA, while the CaSO 4 and β-TCP scaffold performed marginally better. The L-RSF scaffold showed expression levels higher than these ceramic scaffolds and the M-RSF scaffold outperformed all the scaffolds by at least 2x to 3x expression levels.
These results suggest that SF is a superior material for bone void filling applications. Further, the structural attributes of the scaffold such as bulk porosity, pore size, and compression modulus also significantly influence the performance in bone void filling applications.