zinc doped brushite and silk scaffolding in

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Tissue Engineering and Regenerative Medicine

Synergistic effects of silicon/zinc doped brushite and silk scaffolding in augmenting the osteogenic and angiogenic potential of composite biomimetic bone grafts Joseph Christakiran Moses, Mainak Dey, K. Bavya Devi, Mangal Roy, Samit Kumar Nandi, and Biman B. Mandal ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01350 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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ACS Biomaterials Science & Engineering

Synergistic effects of silicon/zinc doped brushite and silk scaffolding in augmenting the osteogenic and angiogenic potential of composite biomimetic bone grafts Joseph Christakiran Moses1, Mainak Dey 2, K. Bavya Devi 3, Mangal Roy 3, Samit Kumar Nandi 2, * and Biman B. Mandal 1, * 1Biomaterial

and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering,

Indian Institute of Technology, Guwahati-781039, Assam, India 2Department

of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery

Sciences, Kolkata-700037, West Bengal, India 3Department

of Metallurgical and Materials Engineering, Indian Institute of Technology Kharagpur,

Kharagpur-721302, West Bengal, India

*Corresponding

authors:

Biman B. Mandal E-mail: [email protected]; [email protected] Tel: +91-361-258-2225 Fax: +91-361-258-2249 Samit Kumar Nandi E-mail: [email protected]; [email protected]

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ABSTRACT Cell instructive scaffolding platforms displaying synergistic effects by virtue of their chemical and physical cues have tremendous scope in modulating cell phenotype and thus improving the success of any graft. In this regard, we report here the development of Si and Zn doped brushite cement composited with silk scaffolding which hierarchically emulated the cancellous bone. The composite scaffolds fabricated exhibited an open porous network capable of enhanced osteoblast survival as attested by increased alkaline phosphatase activity and also sustaining osteoclast activity affirmed by tartrate resistant acid phosphatase staining. Moreover, the chemical cues presented by dissolutions products from the composite scaffold enabled the osteoblasts to secrete proangiogenic factors which favoured better endothelial cell survival, confirmed through in vitro experiments. Moreover, the efficacy of these composite biomimetic scaffolds was validated in vivo in volumetric femur defects in rabbits which revealed that these matrices influenced vascular cell infiltration and favoured the formation of matured bony plate. Fluorochrome labelling studies and micro-tomography analysis revealed that at the end of three months, the implanted composite scaffolds had completely resorbed leaving behind neo-osseous tissue vouching for clinical translation of these composite matrices as viable and affordable bone graft substitutes. Keywords: biomimetic, proangiogenic, bone tissue engineering, resorbable, silk fibroin, brushite cement, doping;


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1. INTRODUCTION One of the unfathomable quests in the domain of regenerative medicine is to attain the best possible regeneration of defective tissue identical to the healthy tissue in all aspects and complete restoration of adjoining wound areas after surgical implantation. Tissue engineering approaches are constantly optimized, reiterated and scrutinized to achieve the ideal strategy which can be easily deployed in such demanding clinical settings. With this perspective, bone tissue engineering in the last decade has seen incremental upgrade to strategies for repair of critical sized defects, fractures and non-unions which include use of osteogenic factors, proangiogenic factors, engineered stem cell based approaches and even the application of smart third generation smart composites. Howbeit, the biggest challenge that still beleaguers clinicians is the avascularity or inability to initiate neoangiogenesis in implanted bone grafts 1. The native bone tissue consists of dense vasculature made up of macrovessels branching into fine bed of microvessels and capillaries which regulate the bone homeostasis and fracture healing by homing mononuclear cells, vascular precursor cells, pericytes and stem cells. When a graft is implanted in a defect site, the graft must facilitate the integration of these vascular networks through anastomosis and through sprouting of new blood vessels within the graft, which determines its success after implantation 2. Several attempts addressing this vasculature conundrum have come up in the recent years such as use of pre-vascularized constructs where endothelial cells are co-cultured with stem cells, microprinting or patterning and delivery of vasculogenic factors 3. In contrast, a more facile approach to facilitate spontaneous angiogenesis and subsequent remodelling involves the use of certain metallic ions which help in stabilizing the hypoxia inducible factor (HIF-1α) 4. In this context, calcium phosphate cements (CPCs) have been extensively investigated for substitutions with metals such as Mg, Sr, Si, Zn, Li, Fe and Ag to impart special features such improved osteogenesis, osseointegration, magnetic and antimicrobial properties 5. It is also pertinent to note that CPCs are the most commonly used bone cements for stabilizing prosthetic load bearing implants owing to their mouldability, low setting times, excellent dissolution and resorption post implantation 6. Among the CPCs, brushite cements 3 ACS Paragon Plus Environment


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(BrCs) have carved a niche of their own because of their bioactivity and ability to resorb at physiological conditions unlike apatite base cements 7. Cellular fate is greatly influenced by its microenvironment and osteoblasts in particular are very sensitive cells that respond to changes in matrix stiffness, ionic gradients and surface roughness8. Therefore, it becomes all the more crucial to develop scaffolding matrices which could harness these physico-chemical cues to modulate osteogenic cell physiology. Driven by the demanding constraints set by clinicians to look for alternatives that can best match the autogenic or allogenic bone grafts, composite bone substitutes have shown some promise in the recent years. In this regard, silk fibroin (SF) as scaffolding matrix has garnered much attention owing to its ease in aqueous processing, amenability to be deployed into many formats, minimal immunogenicity, cost-effectiveness, superior cell supportiveness, and capability to tailor the mechanical and degradation profiles

9-10.

Taking

incentives from SF, suitable cell instructive scaffolding matrices can be easily fabricated which could serve as an ideal matrix for bone regeneration. To this end, we have investigated the effects of Si and Zn doping on setting time and mechanical properties of BrC

6

and also looked into the use of silk scaffolding matrices as osteoconductive

substrates 11-12. However, the feasibility of doped (Si or Zn) BrC in modulating cellular fate relating to osteogenesis and angiogenesis have remained unexplored. This led us in integrating the beneficial properties of SF and BrC in developing biomimetic composite scaffolding matrices for bone tissue engineering applications. To put the current work in perspective, the study delineates the synergistic effects of these composites viz. (i) the effect of doping Si or Zn or binary doping (Zn and Si) on BrC on osteoblasts, osteoclasts and endothelial cells; (ii) the effect of resorbable silk scaffolding (derived from mulberry SF Bombyx mori) in serving as an osteoconductive matrix. In order to understand these two aspects, we have characterized the composite matrices physico-chemically, conducted in vitro biological experiments and eventually validated the efficacy of these matrices in repairing volumetric bone defects in rabbits.


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2. METHODOLOGY BrC and Si, Zn and Si/Zn dual doped BrC were prepared following our previously published protocols (Detailed methodology provided in supporting information file). Similarly, B. mori SF was isolated from mulberry cocoons following our established lab protocols 13-14. Degummed SF fibers obtained were used to fabricate the composites by mixing 1:1 (wt/wt) ratio of silk microfibers with brushite cement and packed into Teflon moulds (Figure 1A). The packing density and fabrication of reinforced silk scaffolding matrices were optimised in our previous studies 11, 14. Detailed description of the synthesis, isolation and fabrication are provided with great detail in supporting information file. The freeze-dried constructs used in the study are listed in Table-1.

Scaffold

SF BrC/SF Si-BrC/SF Zn-BrC/SF Zn+Si-BrC/SF

Table 1. Scaffolds used in the study Silk fibroin Silk Brushite Scaffolding nature solution fibroin Cement (per mL) microfiber Used 2 % (wt/v) 40 mg Silk fiber reinforced matrices B. mori without brushite cements 2 % (wt/v) 20 mg 20 mg Silk fiber reinforced matrices B. mori BrC with BrC brushite cement 2 % (wt/v) 20 mg 20 mg Silk fiber reinforced matrices B. mori Si-BrC with Si-BrC brushite cement 2 % (wt/v) 20 mg 20 mg Silk fiber reinforced matrices B. mori Zn-BrC with Zn-BrC brushite cement 2 % (wt/v) 20 mg 20 mg Silk fiber reinforced matrices B. mori Zn+Si-BrC with Zn+Si-BrC brushite cement

The fabricated scaffolds were physico-chemically characterized using field emission scanning electron microscope (FESEM) (∑IGMA, Carl Zeiss, Germany) equipped with energy dispersive Xray analysis (LEO 1430VP®, Oxford Instruments, UK), X-ray diffractometer (XRD) (Rigaku TTRAX III, Japan), infrared spectrometer (Shimadzu, Model: IR Affinity-1S WL), Universal Testing Machine (UTM, Instron 5944, U.S.A.). In vitro biological studies were carried out using human osteosarcoma cell line (MG63), human monocytes (THP1 cells) differentiated to osteoclasts and primary endothelial cell isolated from 5 ACS Paragon Plus Environment


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porcine descending aorta following our earlier protocols. Osteogenic potential was checked by assessing the viability using calcein-AM (stains live cells green) /ethidium homodimer (stains dead cells red), cellular proliferation using alamar blue assay and biochemically measuring alkaline phosphatase (ALP) activity. Angiogenic potential was assessed through collagen gel tube formation assay, proliferation using alamar blue assay, propidium iodide staining to assess viability using flowcytometry and biochemical estimation of nitric oxide (NO). Osteoclast activity using tartrate resistant acid phosphatase (TRAP) staining was assessed on the composite scaffolds. Detailed descriptions of these in vitro biological studies are provided in supporting information file. In vivo bone forming ability was assessed to evaluate the volumetric bilateral defect healing in epiphyseal femurs of mature New Zealand white rabbits. The animal experiments were carried out in accordance to the guidelines which were reviewed and approved by the Institutional Animal Ethical Committee (IAEC), West Bengal University of Animal and Fishery Sciences (WBUAFS), West Bengal, India (Approval No. Pharma/IAEC/166 dated 1.12.2015). For the study, 36 rabbits of either sexes each weighing ~1.8 kg were used. After 1 and 3-months post-surgery, animals were euthanized and the distal femurs were harvested. They were subsequently characterized through histological examination, fluorochrome labelling and micro-computed tomography (Phoenix V|tome|xs, GE; Germany). Detailed descriptions of these methodologies are provided in supporting information file. 3. RESULTS 3.1 Morphological and elemental analysis of composite scaffolds The composite matrices fabricated through freeze drying process resulted in obtaining a robust sponge like scaffold as depicted in the scheme (Figure 1A). The micro-architectural analysis was undertaken through FESEM analysis and all the matrices presented a porous, open and interconnected network (Figure 1 B-F). The silk microfibers used in the current study acted as the surface on to which the brushite cement and other doped brushite cement got functionalized as seen in Figure 1 C, D, E and F. The introduction of brushite cement variants did not drastically vary the pore size distribution, as 6 ACS Paragon Plus Environment

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all the scaffolds exhibited pore sizes ranging from 50 – 200 μm (Figure 1 G) in comparison to control silk matrices (Figure 1 B), which is in correlation with our previously published reports exhibited pore sizes of similar range11, 15-16.

Figure 1. A i) Scheme representing the methodology for fabricating (control) silk fibroin (SF) scaffolds and A ii) scheme representing the methodology for fabricating experimental silk doped brushite cement composite scaffolds; Elemental mapping reconstructed from the field emission electron micrographs for the different composite scaffolds; B) SF, C) BrC/SF, D) Si-BrC/SF, E) ZnBrC/SF, F) Zn+Si-BrC/SF and G) pore size distribution analysed from electron micrographs. 7 ACS Paragon Plus Environment


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The presentation of open fibrous network is essential for cellular infiltration, migration of vascular cells, immune cells which facilitate the initial graft recognition and further remodelling of the scaffold 17.

The presentation of biophysical cues such as stiffer or relatively rougher surface has been seen to

maintain the osteogenic phenotype

18.

Interestingly, the incorporation of silk fibers increases the

surface area for cell attachment, which is crucial for osteoblast cell survival within the scaffold. The incorporation of brushite cement functionalisation was confirmed by elemental analysis through energy dispersive X-ray spectrometry equipped in FESEM. The experimental stoichiometric composition obtained for the BrC cement synthesized and subsequently used for scaffold fabrication was in accordance to the theoretical doping percentages (Supporting information: Figure S1). In BrC/SF cement, Ca/P stoichiometric ratio of ~1.5 confirming the un-doped β-TCP presence (Figure S1); in Si-BrC/SF the presence of 0.5 (wt %) doping of Si; in Zn-BrC/SF the presence of ~0.25 (wt%) doping of Zn; and in Zn+Si-BrC/SF the presence of ~0.5, ~0.25 (wt%) doping of Si and Zn respectively was affirmed. Moreover, the presence of these brushite cement granules increased the surface roughness as visualized from the electron micrographs in addition of serving as chemical cues for triggering various cellular downstream processes pertaining to osteogenesis. 3.2 Compositional analyses of composite scaffolds Compositional analyses of the fabricated composite silk matrices were carried using FTIR and XRD analyses to ascertain their chemical functionalities. Infrared spectroscopy revealed the functional groups pertinent to the SF and BrCs used for fabrication had amalgamated in producing the composite scaffolding matrices (Figure 2 A). Characteristic peaks for SF were observed at 1650 - 1600 cm-1 indicating presence of amide-I band denoting C=H stretching, 1550- 1500 cm-1 denoting C=N stretching of amide-II band and 1260 - 1210 cm-1 implying amide-III band corresponding to C-N stretching 19. In addition to the SF’s peaks, signature peaks for β-TCP and calcium phosphates present in brushite cements were noticed as peaks denoted by d, e and f at 1046 cm-1, 895 cm-1, and 530 cm1

respectively depicting asymmetric and symmetric stretching of P-O and bending of O-P-O of

phosphate groups of calcium phosphates20-21, as shown in Figure 2 A. Moreover, in the composite 8 ACS Paragon Plus Environment

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matrices a blue shift in amide-I peak (a shift from ~ 1621 cm-1 to ~ 1656 cm-1) was noticed (Figure 2 Bi) which is witnessed during β-sheet transitions in silk matrices22 possibly due to the interactions of calcium groups of BrC with carboxylic groups of SF side chains and rendering more β-sheet conformation. This hypothesis was affirmed when amide-I spectra of SF and brushite composite group (BrC/SF) were deconvuluted (Figure 2 B ii and iii) wherein an increase in β-sheet content was noticed in BrC/SF in comparison to SF. Table 2 Crystallinity, d-spacing and crystallite size of silk matrices used in the study Sample

Diffraction angle (2θ) 17.35° 20.35°

d spacing (nm) 0.531 0.489

Crystallinity Xc (%) 15.2 18.3

Crystallite size D (nm) 8.89 13.67

BrC/SF

25.86° 31.24°

0.201 0.149

69.3 76.4

98.23 57.26

Si-BrC/SF

25.86° 31.24°

0.213 0.159

68.3 75.4

102.68 59.26

Zn-BrC/SF

25.86° 31.24°

0.216 0.156

70.2 77.3

105.14 60.03

Zn+Si-BrC/SF

25.86° 31.24°

0.227 0.163

73 76.2

101.23 61.57

SF



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Figure 2. Compositional analyses ascertained through A) Fourier infrared spectra B) i) spectra showing blue shift in SF vs. BrC group deconvuluted Amide-I spectra for i) SF and ii) BrC/SF and C) X-ray diffractograms of different scaffolding groups used in the study; Uniaxial unconfined compressive mechanical studies on fabricated scaffolds; D) representative stress-strain curves for different scaffolds; E) Young’s modulus and F) compressive stress at 80% strain for different scaffolds used in the study; G) Unconfined cyclic mechanical assessment of the composite matrices; their compressive stress vs. time plot (# represents significant difference at p≤0.05 and $ represents significant difference at p≤0.01) Phase identification and the crystallinity of the fabricated composite silk matrices were undertaken using wide angle X-ray diffractometer (Figure 2 C). The SF scaffold exhibited two broad amorphous shoulder peaks a and b typical of the β-sheet structures noticed in its chemical conformation (silk II conformation

23-24)

as a result of β-sheet induction through ethanol treatment and the native

degummed chopped silk fibers used here as reinforcements, respectively. The amorphous peaks of SF were subdued by crystalline peaks of the calcium phosphates from the brushite cements in the composite scaffolds. Of interest is the characteristic peaks of β-TCP (2θ values for 25.86°, 28.20°, 10 ACS Paragon Plus Environment

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31.24°, 53.32° corresponding to their miller indices (1 0 10), (2 1 4), (0 2 10), (2 0 20)) and dicalcium phosphate dehydrate (DCPD) (2θ values for 35.75°, 40.20° corresponding to their miller indices (0 2 2), (1 5 2)) which were noticed from the diffractograms 6, 25-26. The crystal parameters for signature peaks of SF and β-TCP were calculated (Table 2) to assess whether the doping or composite formation had any effect on crystallinity or crystal size. No notable changes were seen for crystallinity, however the crystallite size (D) and d spacing increased in doped variants as compared to the doped variants, possibly due to the crystal parameter changes as result of substitutions in the β-TCP lattice structure. 3.3 Compressive mechanical studies on the composite scaffolds Unconfined uniaxial compressive testing revealed all the samples tested exhibited sponge-like behaviour, with similar stress-strain behaviour (Figure 2D) noticed for all the groups. All the groups exhibited three discrete regions (Figure S2 A, Supporting Information), an initial linear region corresponding to the elastic region (0 % to 10 % strain - from which the Young’s modulus was calculated), followed by a collapse plateau region (15 % to 40 % strain) and then subsequently a densification region (> 50 % strain). The plateau region corresponds to the scaffold yielding due to internal pore bending and the final densification region corresponds to pore buckling and subsequent bulk densification of the sponge

27.

In comparison to SF, the incorporation of brushite cements

noticeably increased the Young’s modulus (Figure 2E) with increasing order as follows BrC/SF > Si-BrC/SF > Zn-BrC/SF > Zn+Si-BrC/SF. BrC/SF exhibited ~2.10-fold increase in Young’s modulus in comparison to SF while Si-BrC/SF exhibited ~1.89-fold increase. The incorporation of zinc in the brushite cement was found to decrease the compressive stress (measured at 80% strain) and the Young’s modulus in Zn-BrC/SF group (Figure 2F). A similar observation was previously reported wherein the effect of incorporation of zinc as dopant in brushite cements significantly decreased the compressive strength 6. Cyclic compression studies revealed all the scaffolds mostly retained their elastic behaviour (Figure S2 B-F). The hydrated scaffolds were compressed for 20 cycles to 50 % of their origin size. It was noticed from that for the first 4-5 cycles a slight deformation was noticed but 11 ACS Paragon Plus Environment


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however for the remainder of the cycles there was no inelastic deformation noticed. As evident from the uniaxial mechanical testing experiments, the scaffolds exhibited superior mechanical resilience in the following order BrC/SF > Si-BrC/SF > Zn-BrC/SF and Zn+Si-BrC/SF (Figure 2G). 3.4 In vitro biological studies 3.4.1 Osteogenic and osteoclastogenic potential of the composite scaffolds The cellular viability in the MG63 cell seeded constructs were assessed using calcein-AM staining wherein the bright green fluorescence represent the presence of viable cells (Figure 3A) while the innate red autofluorescence of SF based scaffolds served to identify the cells within the matrices (Figure S3, Supporting Information). All the scaffolding matrices supported the seeded cells with no matrix exhibiting any noticeable toxicity as inferred from the absence of dead cells (stained red with ethidium homodimer). The presence of fibers (Figure 3Ai) enhanced the available cell surface area for attachment in the silk scaffolding matrix in addition of serving to improve the mechanical properties. The cells appeared to take a more rounded morphology in the 3D matrix as opposed to spindled morphology observed in 2D tissue culture plate (Figure 3Avi). The viability of the cells was quantitatively assessed using alamar blue assay (Figure 3B) and it was found that cells proliferated within the matrices as compared to day-1. The silk matrices supported the growth of cells better evident by the better performance in comparison to other brushite silk composites. Si-BrC/SF and BrC/SF performed better than the Zn-BrC/SF < Zn+Si-BrC/SF groups. The plausible reason could be that these osteoblast-like cells are acquiring a functional phenotype maturing towards a mineralised state. MG63, though an osteosarcoma cell line consist of a heterogeneous cell population of mature and immature osteoblasts 28 and the presence of BrC enable in triggering the switch from immature to mature phenotype.


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Figure 3. Osteogenic and osteoclastogenic potential of fabricated composite scaffolds; A) Live/dead cell imaging using calcein-AM (stains live cells green) and ethidium homodimer (stains dead cells red) for i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) MG63 cells on tissue culture plate (with insets of higher magnification); B) cellular viability using alamar blue assay; C) alkaline phosphatase activity assessment of the cell seeded constructs; D) tartrate resistant acid phosphatase (TRAP) staining for i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) osteoclasts differentiated on tissue culture plate (# represents significant difference at p≤0.05 and 13 ACS Paragon Plus Environment


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$ represents significant difference at p≤0.01); (multinucleated osteoclasts are represented with green arrows). To establish this relation, we further estimated the alkaline phosphatase activity (Figure 3C), a key mediator enzyme involved in mineralisation process. Interestingly, we found that Zn-Br/SF and Zn+Si-BrC/SF exhibited higher ALP activity than the other groups, confirming that incorporation of Zn and Si as dopants enabled in improving the osteogenic potential of the composite scaffolds. Furthermore, to assess whether the doping of ions had any effect on the osteoclast activity, the osteoclastic differentiation potential of human monocytes was studied on the composite matrices. After 14 days of differentiation protocol, the cell laden constructs were histologically processed and stained for tartrate resistant acid phosphatase (TRAP). TRAP is a key enzyme secreted by active osteoclasts which after staining appear as purplish to dark granules in the cytoplasm (Figure 3 Dvi). Interestingly, the osteoclast activity was not hampered by any means and relatively same densities of multinucleated osteoclasts (represented by green arrows) were noticed in all the groups, vouching for the resorption effectiveness of these developed scaffolds (Figure 3 Di-v). 3.4.2 Angiogenic potential of the composite scaffolds Addition of certain dopants in ceramics has been proven to confer multifunctionality to the ceramics such as enhancing the osteogenesis, providing antimicrobial traits, enabling better remodelling or resorption

29-30.

Here we tried to assess the angiogenic potential of the composites and tried to

decouple the effect of dopants on osteoblast like MG63 cells to trigger angiogenic signalling. Hence the conditioned media from the MG63 cell seeded constructs were taken to assess the tube formation in vitro using collagen gels (Figure 4A). In comparison to SF, BrC/SF groups (Figure 4Ai and ii) the conditioned media from doped BrC/SFgroups (namely Si-BrC/SF, Zn-BrC/SF, Zn+Si-BrC/SF) (Figure 4Aiii-v) enabled in better tube formation. The tubes formed were quantified using Image-J analysis and it was observed that Si-BrC/SF and Zn-BrC/SF significantly increased the number of nodes and tubes as opposed to SF and BrC/SF matrices, suggesting the doping supported better angiogenic performance (Figure 4B-C). 14 ACS Paragon Plus Environment

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Figure 4. Angiogenic potential of fabricated composite scaffolds; A) tube formation assay using calcein-AM stained porcine endothelial cells for conditioned media obtained from cell seeded constructs of i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF; vi) tube length and vii) number of tubes and nodes noticed per imaging field from the fluorescent micrographs; B) viability of seeded endothelial on silk matrices i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, viii) percentage of dead cell population; C) proliferation of endothelial cells assessed by alamar blue assay and D) biochemical estimation of endothelial nitric oxide (NO) (# represents significant difference at p≤0.05) 15 ACS Paragon Plus Environment


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To further evaluate the angiogenic performance of the fabricated scaffolds, endothelial cells were seeded on to the matrices and supplemented with conditioned media from osteoblast laden scaffolds and assessed for viability, proliferation and functionality. The endothelial cells grown on tissue culture plate supplemented with endothelial cell media served as positive control. In comparison to control, endothelial cells within the composite matrices exhibited lower proliferation rate (Figure 4C). Between the doped BrC groups (Si-BrC/SF, Zn-BrC/SF, Zn+Si-BrC/SF) no significant difference was found, however these groups performed better than BrC/SF. To quantitatively infer whether lower proliferation is correlated with cell death, the viability of endothelial cells harvested from scaffolds at day-7 through flow-cytometry. Propidium iodide stains dead cells and it was observed within the scaffolds, ~7 to 8% dead population was noticed comparable to control (Figure 4B). Though the scaffolds supported endothelial cells, we evaluated their functionality by biochemical estimation of nitric oxide. Endothelial NO is a key regulator of vascular cell physiology and it was noticed that the doped BrC groups (Si-BrC/SF, Zn-BrC/SF, Zn+Si-BrC/SF) exhibited increased NO as compared to control and BrC/SF group (Figure 4D). This substantiated the findings from collagen tube formation assay where increased number of tubes and sprouts were noticed in doped BrC groups in comparison to other groups. 3.5 In vivo biological studies Based on the in vitro physico-chemical and biological validation of the developed composited we further evaluated the potency of these composites to serve as multifunctional bone graft substitutes in volumetric bone defect created in distal epiphyseal rabbit femurs. For the study, skeletally mature New Zealand white rabbits were taken as model animals. After implantation of the composite grafts post-surgery, the animals were closely monitored during post-operative care. During the period of investigation, the animals exhibited neither any signs of inflammation or associated effects nor were found to exhibit lameness, disability and deterioration of movement. The femurs at 1 month and 3 months post-surgery were harvested after euthanasia and the experimental groups showed at evidences of healing of defect regions with smooth bony exterior cover over experimental groups 16 ACS Paragon Plus Environment

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(Figure S4, Supporting information) in particular the Zn+Si-BrC/SF groups. In comparison, the empty defect group and SF group minimal bony growth was noticed where dents of defects created were visible even at 3-months extracted femurs. 3.5.1 Histological examination Histological examinations were carried out in order to understand the host-implant interaction and the cellular events that occur during the bone remodelling and regeneration processes involving the orchestrated synchrony of osteoblastic and vascular cells. Figure 5 A i-vi shows the histological evaluation of cellular activity at 1-month post retrieval of femur segments at the bone-implant interface. At 1 month, the control (Figure 5 Ai) (empty defect) exhibited patchy medullary lamination with scanty blood vesicular bridges. The cellular infiltration was noticed to be less and the adjoining medullary cavity contained red blood cells (RBCs), few fat cells and engrossed by fibrous tissue typical of callus induction. Figure 5 Aii, the BrC/SF group showed a relatively better osteogenic structure with Haversian canal and bony lamina commencement. Few regions of the bony plate were seen to be invaded with few osteoblasts and angioinvasion was scanty. Medullary space had deposition of fibrinous mass and slight cortical structuration. Figure 5 Aiii, the Si-BrC/SF group exhibited bony plate containing osseous mass with relatively higher Haversian canal than SF group. Invasion of bony plate with osteoclasts, typical of remodelling phase to form the permanent bony lamina was noticed. The medullary cavity contained few fibrinous cells, few fat cells and was infiltrated with few mononuclear cells. Figure 5 Aiv, the Zn-BrC/SF exhibited relatively wellorganized bony plate compared to SF, Si-BrC/SF and it contained relatively higher and sufficient number of Haversian canals with solid laminar structure. The medullary cavity was found to contain numerous mononuclear cells and RBCs with few fat cells. The angioinvasion characterized by fibrovascular cell infiltration was quite satisfactory compared to other groups indicating bone regeneration. Figure 5 Av, the Zn+Si-BrC/SF group depicted formation of osteogenic tissue and formation of laminar bone and neo-angiogenesis. Fibro-collagenous deposition was quite satisfactory and the 17 ACS Paragon Plus Environment


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medullary cavity contained numerous RBCs, mononuclear cells and fat cells. While Figure 5 Avi, the SF group showed bony structure depicting a nascent cortical plate with medullar matrix formation and the medullary space contained RBCs, fibrous cell infiltration, few osteoblasts with scanty angiogenesis. At 3 months, the control group (empty defect) (Figure 5 Bi) showed discontinuous defect margins and the bony plate were invaded with few osteoclastic and osteoblastic cells. The medullary cavity contained fat droplets, RBCs, few mononuclear cells and onset of scanty angiogenesis was noticed. Figure 5 Bii, the BrC/SF group revealed presence of calcified matrix with bony plate containing numerous Haversian canals sinusoidal space. The lacunae were however blunt and contained very few mononuclear cells. Figure 5 Biii, the Si-BrC/SF group showed bony structure with relatively better Haversian network. Evidences of periosteal restoration were noticed with scanty collagenous fibers. The angio-invasion was moderate and distributed sparsely throughout the neoosseous tissue space. Figure 5 Biv, the Zn-BrC/SF group exhibited compact bony structure with several Haversian canals and interlacunar space. The angio-invasion was relatively better and the medullary cavity contained scanty fibrous space filled with few RBCs and few fat cells. Figure 5 Bv the Zn+Si-BrC/SF showed total periosteal restoration with peri-osseous calcified matrix formation complete with neoosteogenic lamina structure invaded with few osteoblasts. The peri-osseous space contained numerous osteoblasts, osteocytes and mononuclear cells. Figure 5 Bvi, the SF group showed bony structure containing fibro-cartilaginous mass with the medullary cavity filled with fat cells and few mononuclear cells. The medullary structure was invaded with mild neo-osseous tissue.


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Figure 5. In vivo evaluation of bone formation using histological examination of implanted scaffolds i) empty defect (control), ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) SF and fluorochrome labelling assessment vii) empty defect (control), viii) BrC/SF, ix) Si-BrC/SF, x) ZnBrC/SF, xi) Zn+Si-BrC/SF, xii) SF at A) 1 month and B) 3 months post-implantation (Bright yellow region represents new bone (NB) regions whereas green regions represent old bone (OB) areas). From the in vitro results we had noticed that composite scaffolds with dopants (Si-BrC/SF, ZnBrC/SF, Zn+Si-BrC/SF) helped in promoting better endothelial behaviour. To ascertain it further, we evaluated the expression of hypoxia inducible factor, HIF-1α a crucial transcription factor involved in activating proangiogenic signalling and bone sialoprotein (BSP), an important osteogenic marker (Figure 6). At 1-month, post-implantation scaffold groups Si-BrC/SF, Zn-BrC/SF and Zn+Si-BrC/SF expressed HIF-1α (stained red) as opposed to control (empty defect), BrC/SF and SF scaffolding groups. Scanty ingrowth of neo-osseous tissue (stained green for BSP) was observed in control and SF group. Zn-BrC and Zn+Si-BrC/SF showed the better osteogenic performance than the other groups with high cellular infiltration indicative of dense peri-osseous calcified matrix.



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Figure 6. Immunohistochemical assessment for expression of hypoxia inducible factor, HIF-1α (stained red) and bone sialoprotein, BSP (stained green) in different groups at 1 month, A) empty defect (control), B) BrC/SF, C) Si-BrC/SF, D) Zn-BrC/SF, E) Zn+Si-BrC/SF, F) SF, with nuclei counterstained with Hoechst 33342 (blue). 3.5.2 Fluorochrome labelling analysis Fluorochrome labelling studies helps us in understanding the extent of new bone formation and thereby enables us to quantify the bone mineralisation at a particular time point effectively. The fluorochrome OTC labelled new bone which fluoresces with a golden yellow hue and the old bone that fluoresced with a darker green hue was analysed for percentage of new bone formation from the fluorescent micrographs (Figure 5A vii-xii and Figure 5B vii-xii) and is presented in supporting information Figure S5 A and B, Supporting Information. At 1 month, the harvested bone sections showed mild new bone formation (19.2 ± 1.9%) in empty defect group (Figure 5 Avii) whilst in experimental groups BrC/SF and Si-BrC/SF showed relatively more new bone formation (~35%) with majority of golden yellow hue noticed in central region while dark green toned old bone in the periphery (Figure 5 Aviii and ix). Zn-BrC/SF groups showed new bone formation percentage of ~38%, while Zn+Si-BrC/SF showcased the most with 47.5 ± 2.1% new bone formed amongst all the groups (Figure 5 Ax and xi). The SF group (Figure 5 Axii) showed moderate new bone formation of ~ 29%, consistent with histological examination. At 3 months, the amount of new bone formation 20 ACS Paragon Plus Environment

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was noticed to increase in all the sampling groups in comparison to month-1 status. The Zn-BrC/SF and Zn+Si/BrC/SF groups showed extensive bone formation of ~73 % (Figure 5 Bx and xi) in comparison to defect group which only achieved ~ 41% (Figure 5 Bvii). The SF group also showed relatively lesser bone formation of ~49% with new bone areas (golden yellow hue) appearing scattered throughout (Figure 5 Bxii).

Figure 7. Radiographic 2D images of defect bone sites implanted with i) empty defect (control), ii) BrC/SF, iii) Si-BrC/SF, iv)Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) SF; 3D Micro-CT images of defect bone sites implanted with vii) empty defect (control), viii) BrC/SF, ix) Si-BrC/SF, x) Zn-BrC/SF, xi) Zn+SiBrC/SF, xii) SF showing extent of healing after A) 1month and B) 3 months (Red ring showing defect site; yellow arrow showing new bone regeneration) 3.5.3 Micro-computed tomographic analysis The micro-CT analyses offers a more visual understanding of the progression of acquiescence of the composite scaffolds implanted in the defect site and the neo-osseous tissue formed replacing the scaffold matrix (Figure 7). The calcified bone tissue and adjoining native bone appear as white network while the organic and relatively radiolucent composite matrices appear as lesser dark grey entity in the scans. It is evident from the images that neo-osseous tissue formed was in accordance to the porous architecture of the implanted scaffolding matrix, wherein the open porous network enabled 21 ACS Paragon Plus Environment


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cellular infiltration and allowed the osteoblast and vascular cells to lay down the mineralised matrix. At 1 month, the 2D images (Figure 7 Aii-vi) showed evidences of dissolution of matrices implanted with onset of irregular calcified tissue formation noticed in Zn-BrC/SF and Zn+Si-BrC/SF groups (Figure 7 Aiv-v). The 3D images (Figure 7 Aviii-xi) showed that the implanted composites are resorbed into the surrounding bony tissues substantiating that osseointegration of the implanted scaffold groups Si-BrC/SF, Zn-BrC/SF and Zn+Si-BrC/SF was successful (Figure 7 Aix-xi). Conversely, the defect ridges were very much prominent in empty defect group and SF group (Figure 7 Avii,-xii), where the mineralisation onset has not yet begun. At 3 months, the BrC/SF and SiBrC/SF (Figure 7 Bii,iii) showed continued bone regeneration with irregular calcified bony regions whereas a near complete defect cover was noticed in Zn-BrC/SF (Figure 7 Biv) and a complete closure of the defect site was witnessed in Zn+Si-BrC/SF group (Figure 7 Bv). The defect margins were still incomplete in the empty defect (Figure 7 Bi) and scantily clad in SF group (Figure 7 Bvi). The released Si and Zn ions have played a significant role in enhancing the bone regeneration in case of the experimental groups’ faster bone formation. From the 3D images (Figure 7 B iii-v) relatively much finer and matured bone tissue was noticed with higher amount interconnected network similar to woven bone was observed in Zn-BrC/SF and Zn+Si-BrC/SF groups as opposed to BrC/SF groups (Supporting information; Figure S5C). Moreover, the released dopants Si/Zn were completely embedded inside the surround native bone tissue resulting in new tissue invasion with complete periosteum formation and no remnants of the scaffolds were noticed attesting the completely resorbable nature of these developed matrices. Alternatively, in case of empty defect control group (Figure 7 Bi) and SF group (Figure 7 Bvi) the newly formed bone tissue were irregular and less dense while the osseous tissue accumulation was predominant around the defect margins rather on the inside, thus partially covering the defect site. 4. DISCUSSION Over two decades ago a novel class of calcium phosphate based ceramic cements were discovered by Mirtchi et al whose unique proposition was its ability to resorb under physiological conditions31, 22 ACS Paragon Plus Environment

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unlike the most commercially successful hydroxyapatite based cements. These BrCs mainly constituted of acidic monocalcium phosphate monohydrate and basic β-TCP, result in forming a mouldable paste with quick setting properties amenable for bone reconstruction procedures. Over the years, the drawbacks associated with BrC based bone cements such as low mechanical strength, its quick setting times have been tried to be overcome by utilising additives such as pyrophosphates, sulphates, polymers 7, 32. However, the use of such additives greatly compromises the bioactivity and resorbability of BrC. For instance, use of citric acid as an additive in BrC though enhanced the setting time but was seen to reduce the BrC dissolution

33.

However, a more viable alternative is use of

dopants wherein ions such as Sr, Mg, Zn, Si have been shown to modulate the setting time of BrC without hampering its bioactivity. In this regard, we have reported earlier the development of such doped BrC wherein we have modulated the setting time and also the mechanical properties of the brushite cements 6. Doping or ion substitutions garners simplistic yet assuring approach of bringing about profound difference in tweaking the cellular processes associated with osteogenesis, osteoclastogenesis, vascularization and also provides antimicrobial traits to the developed composites, all the more at an inexpensive contention5. The other alternative approaches for bring about changes in osteogenesis and vascularization involve the use of growth factors such as bone morphogenetic proteins (BMPs), vascular endothelial growth factors (VEGF), insulin like growth factors (IGFs), transforming growth factor (TGF-β) or even platelet rich plasma (PRPs) 34. The use of biologicals or such growth factors have its drawbacks associated such as low shelf life, bioactivity of the included growth factor after release and the cost incurred. Meanwhile, metallic ion substitution over the years has been found to boost the performance of ceramic materials. For instance, silicate substituted calcium phosphate have shown to enhance the bone bonding ability of ceramics, while use of zinc which is an important cofactor for many metallo-enzymes such as ALP and even IGF-2 have been reported to enhance the pre-osteoblast maturation and modulating the local immune response35-37, thereby conferring osteoinductive traits to the resulting ceramic.



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For this study, we chose two ions namely Si and Zn for doping BrC cement formulation based on literature and our previous experiences. Accordingly, the incorporation of Zn resulted in increase in setting time while no changes were noticed for Si doping in brushite cements. Howbeit, use of only BrC as void fillers for bone grafting is highly detrimental due to the exothermic reaction associated with its setting, local increase in pH resulting in osteonecrosis and poor mechanical properties of neat BrC itself. Therefore, to overcome these drawbacks we amalgamated the osteoinductive properties of our BrC cements with the bioactive properties of silk fibroin (SF). B. mori SF has been documented extensively in literature for various tissue engineering applications owing to its multifaceted nature such as unique tuneability in degradation and mechanical properties, facile aqueous fabrication, amenability to format into different formats and superior cell supportiveness 10. Moreover, SF from our previous endeavours has been demonstrated as an excellent scaffolding platform for bone tissue engineering applications due to its osteoconductive nature and ability to resorb inside the implanted site with time

12, 14, 16.

Thus, SF becomes an excellent choice of material to couple with BrC to

augment its bioresorbability, improve its osteoconductivity and mechanical properties without compromising on any of the goodness associated with each individual entity. Besides, the slight basic nature of silk fibroin compensates for the acidic release products which is associated with BrC making the composite thus developed in the study all the more supplementary to each other for its success. The facile fabrication strategy adopted here is in itself very simple and deems fits for easy scale-up. Sometimes the most minimalistic of approaches are the ones which clinicians prefer to be used in clinical setting. The greener all aqueous processing strategy utilised here involved mixing of degummed chopped silk microfibers with the BrC cement to functionalise the fibers. Thus, functionalized fibers acted as reinforcements for the silk scaffolding matrix which was fabricated via freeze-drying process. The fabricated scaffolds exhibited good open porous architecture with good interconnection within pores. Heterogeneous fibrillar pores were seen present in all the scaffolding groups used in the study. The mean pore size of SF scaffolds was found to be ~120 μm while the addition of BrC did not adversely bring down the pore size and porosity of the scaffolding matrices 24 ACS Paragon Plus Environment

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where the least mean pore size of ~ 80 μm was noticed in Si-BrC/SF and BrC/SF while Zn-BrC/SF and Zn+Si-BrC/SF groups largely remained unaffected. Besides it has been reported that pore sizes ranging from 40 to 135 μm to be optimal for osteoblast survival and subsequent infiltration of mononuclear cells and fibro-vascular cells during the bone remodelling phase

38,

whereby these

criteria are fulfilled by the matrices developed here. The appropriate presentation of physico-chemical cues is very important in driving the differentiation fate of stem cells which are very sensitive to surface chemistry. Surface roughness ranging from 0.5 – 4.7 μm have been reported to greatly enhance the osteogenic fate of the stem cells through faster osteogenic commitment and increased extracellular matrix turnover 39. The BrC and doped BrC functionalization of degummed B. mori silk fibers greatly enhanced the surface roughness as seen through the electron microscopy images and from the elemental analysis we confirmed the deposition of these BrC granules over the fibers. The presentation of surface roughness and chemical cues advocates for the two-fold benefits which could potentially drive the osteogenic differentiation of infiltrating stem cells through synchronous pairing. Furthermore, we dwelled into understanding the compositional nature of the developed matrices through XRD and FTIR studies. Phase identification through XRD analysis revealed the two most predominant phases were β-TCP and DCPD and no other contaminating ceramic phases were formed owing to the low amount of the dopants utilised in the study. Furthermore, we analysed the crystal parameters of the composites and found that the doping had little effect on the crystallinity of the BrC, thereby not compromising on its dissolution or resorption kinetics. Reports on highly crystalline calcium phosphates have been always associated with slower dissolution rates (Kd) of the order 10-40 to 10-59 40-41. Other crystal parameters such as d spacing and crystallite size (D) increased in the doped variants

in comparison to undoped BrC. This is ideally because the dopants used in the study i.e. Si4+ and Zn2+ undergo ion substitutions with P5+ and Ca2+ respectively in the β-TCP crystal lattice, thereby an increase in crystallite size and d spacing is noticed to accommodate these dopant substitutions. At the atomic level, each of these substitutions has an effect on the dissolution kinetics of the BrC. For 25 ACS Paragon Plus Environment


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instance, the Zn2+ substitution in Ca2+ lattice points stabilizes the β-TCP structure and hence retards the dissolution 42 while Si4+ for P5+ substitutions creates O2- vacancies which in turn improves the βTCP dissolution 43. Characteristic signature peaks for both SF and calcium phosphates were observed from infrared spectroscopy indicating the successful amalgamation of these two unique entities into a single composite. Interestingly, when compared to the only silk scaffolding matrix (SF), the composites exhibited a blue shift in amide-I region. We deconvuluted the spectra and observed that the total percentage of β-sheet content had increased by ~20 %. This infers that there were possible interactions of calcium or silicate groups of BrC with carboxylic groups of SF side chains resulting in the composites achieving more β-sheet conformation. Interesting to note, silk matrices’ stability and mechanical properties are correlated with its β-sheet content 12-13 and hence the acquiesce of more β-sheet content in the composites predicts the superior mechanical properties deemed to possess than the pristine silk matrices alone. For the success of any bone graft its mechanical compliance is an essential prerequisite. It has been observed that bulk stiffness greater than ~40 kPa favours the osteogenic differentiation of mesenchymal stem cells and we have witnessed the same in our previous reports also

11, 14, 16.

The

presentation of such stiffer matrix modulates the osteogenic commitment and its subsequent mineralised matrix turnover through a mechanostransductory pathway involving Rho kinases (ROCK) and focal adhesion kinase (FAKs)

44.

In our case, the silk matrices exhibited Young’s

modulus (E) of ~40 kPa and we have determined the densities (D) of these matrices to be ~75 kg/m3 and correlating them to a power law equation, Log E = -4.37 + 1.88 log D, for silk particle reinforced scaffolds, these scaffolds is predicted in the same range for power law equation hypothesized for cancellous bone (Log E = -4.10 + 2.47 log D) 14. Thus these matrices’ mechanical strength could be modulated proportionately by increasing the densities through either increasing the reinforcing fiber content or the BrC content, thereby possibly achieving higher compressive strength. Besides, it was noticed that the incorporation of BrC in the silk matrices increased the Young’s modulus by two folds, 26 ACS Paragon Plus Environment

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substantiating the superior mechanical properties of these composite scaffolds. Furthermore, it has been seen the functionalization of the fibers with BrC provided granularity and thus bestowed fatigue resistance during cyclic compression studies by processes similar to composite hierarchical bone involving micro-crack propagation and intra-particular stress shielding effects. The physiology of the osteoblasts in addition to response to these biomechanical cues also is largely influenced by the local chemical milieu. For instance, it has been noticed that the commitment of a differentiating osteoblast can be reprogrammed by addition of soluble induction factors or it can lose its osteogenic commitment over time without appropriate chemical cues 8. Therefore, seeing these dire straits for the need to maintain the long term osteogenic commitment, the incorporation of osteoinductive BrC is very much beneficial. From our preliminary in vitro results utilising osteoblast like cells we tested this hypothesis and it was seen that the incorporation of BrC and doped BrC significantly augmented the osteogenic capacity which was affirmed by the ALP activity assessment. The seeded cells were viable after 7 days with well clustered morphology similar to bone noduling where the onset of mineralization is generally witnessed. The survival of these osteoblasts can be directly related to the open porous network which facilitated better diffusion of nutrients and the biomechanical cues provided by the stiffer matrices which we had discussed earlier. Furthermore, in order to assess whether the incorporation of BrC or doped BrC had any detrimental effect on osteoclastogenesis, we had seeded mononuclear cells and differentiated them into osteoclastogenic lineage. Interestingly, the composite silk matrices showed no adverse effect rather supported better or at least on par with the only silk matrices. This is very much essential because for successful osseointegration of graft, there are two axes to it. One is the ability for bone forming osteoblasts to lay down the matrix, and the other is the multinucleated osteoclasts to resorb the implant and the matrix to make way for regeneration. This synchrony is very much needed as neo-osseous tissue slowly replaces the implanted matrix and composite silk matrix is bestowed with better resorbability due to bioresorbable silk and BrC used in the study. The infiltration of osteoblasts, stem cell homing is greatly dependent on vascularization and it has been seen that the failure of most of the bone 27 ACS Paragon Plus Environment


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implants is due to poor neo-vascularization 1. Our results showed that endothelial cell survived better from the conditioned media of osteoblasts cultured on the doped BrC/SF scaffolds through tube formation. The endothelial cells in the doped variants supported more number of tubes of greater length and more nodding points were also seen from the calcein-AM staining. The functionality in terms of NO secretion by endothelial cells within the doped composite variants (Si-BrC/SF, ZnBrC/SF and Zn+Si-BrC/SF) were also higher. The mechanism underlying is that under normoxia hypoxia inducible factor (HIF-1α) is destined for proteolytic degradation by prolyl-4-hydroxylase enzyme. However, if HIF-1α is stabilised by cationic metal ions they are not prone for ubiquitination but rather couple with HIF-1β to act as transcription factors which trigger hypoxia response elements (HRE) which in turn activates an array of angiogenic factors such as VEGF, erythropoietin and nitric oxide synthase4, 14. This culminates in downstream processes where in a cascade of events more endothelial cells, pericytes and stem cells are homed to the site as chemotactic angiogenic factors influence the neovascularization processes further. Driven by the positive outcomes from our in vitro results we validated our composites in volumetric defects in distal epiphyseal femurs of rabbits. The doped cement variants especially the dual doped system Zn+Si-BrC/SF group outperformed the undoped BrC/SF and SF groups. Based on the histomorphometry analysis we were able to noticed relatively higher vascular infiltration characterized by invading fibro-vascular tissue and mononuclear cells. This could be attributed to the stabilization of HIF-1α and the activation of hypoxia response elements through which better angiogenic response is favoured within these composites. The presence of osteoclast population at 1 month was also adequate substantiating our in vitro findings that these composite matrices enhanced the angiogenic and osteoclastic activity. At 3 months, heightened osteoblastic activity and maturation of lamellar bone with complete repair of defect edges and even restoration of periosteum was noticed in case of Zn+Si-BrC/SF group whereas SF and empty defect groups still had the defect ridges. Similarly corroborating the histomorphometric analysis we found that in micro-CT analysis well matured neoossseous tissue with woven bone like architecture was noticed at 3 months for Zn+Si28 ACS Paragon Plus Environment

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BrC/SF group with no remnants of implanted scaffold. The percentage of new bone formation reiterated through flurochrome labelling studies also revealed that Zn+Si-BrC/SF group had ~70% new bone whereas a dismal ~40% was noticed in SF group. These findings all together point to the fact that the doping of Zn/Si actively favoured increased osteogenesis without dampening the bone remodelling kinetics. However, more holistic studies relating to the molecular mechanisms are needed to understand better the cellular pathways involved in activation, homing of stem cells and endothelial cells which is beyond the scope of the current study. Howbeit, these findings validate the notion that the doped BrC and silk scaffolding synergistically orchestrate osteogenesis and angiogenesis enabling better bone regeneration. 5. CONCLUSIONS Avascularity or inadequate neovascularization is one of the unrelenting reasons for bone graft failures during atrophic or hypertrophic non-unions. Addressing this latent predicament becomes all the more necessary which we resorted using our biomimetic and bioresorbable composite silk scaffolding matrices. We evaluated the use of Si and Zn as dopants to BrC which we successfully composited with B. mori silk fibroin to obtain a much mechanically compliant yet bioactive scaffold. The consensus of this scaffolding can be delineated into two folds: (i) the dopants Si/Zn enabled in triggering angiogenic signalling which is pivotal for bone regeneration and (ii) the silk matrix which provided an osteoconductive milieu favouring osteoblast migration, osseointegration, extracellularmatrix turnover and osteoclast resorption. The chemical cues and biophysical platform synergistically orchestrated the two main cellular events i.e. osteogenesis and angiogenesis in tandem favouring complete regeneration of volumetric bone defects in distal epiphyseal femurs of rabbits with complete restoration of the periosteum. However, the underlying mechanism involving resorption kinetics, cellular responses and the ability of the composites to repair full-thickness defects need to be evaluated exhaustively which is beyond the scope of the current work. Nonetheless, the promising findings from this work vouches for the potential clinical translation of these Si/Zn doped brushite cement silk scaffolding matrices for smart, cell instructive bone tissue engineering. 29 ACS Paragon Plus Environment


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CONFLICT OF INTERESTS There is no conflict of interests SUPPORTING INFORMATION Elemental spectral distribution from FESEM-EDX analysis; cyclic mechanical assessment; extent of new bone formation by fluorochrome labelling and micro-CT analysis. ACKNOWLEDGEMENTS B.B.M. is thankful for the generous funding support from funding agencies, DST and DBT, Government of India. J.C.M is grateful to the Ministry of Human Resource Development (MHRD), Government of India for fellowship. We also appreciate the kind support provided by Mr. Bibhas Kumar Bhunia and Mr. Gopal Pandit for assistance in wide-angle X-ray diffraction. The Central Instrumentation Facility (CIF) and the Centre for the Environment of IIT-Guwahati are also acknowledged for providing high end instrumentation support. SKN gratefully acknowledge the support by the Vice Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata, India REFERENCES 1.

Santos, M. I.; Reis, R. L., Vascularization in bone tissue engineering: physiology, current

strategies, major hurdles and future challenges. Macromolecular bioscience 2010, 10 (1), 12-27. DOI. 10.1002/mabi.200900107 2.

Klotz, B.; Lim, K.; Chang, Y.; Soliman, B.; Pennings, I.; Melchels, F.; Woodfield, T.;

Rosenberg, A.; Malda, J.; Gawlitta, D., Engineering of a complex bone tissue model with endothelialised channels and capillary-like networks. European cells & materials 2018, 35, 335-348. DOI: 10.22203/eCM.v035a23 3.

Fu, J.; Wang, D.-A., In Situ Organ-Specific Vascularization in Tissue Engineering. Trends in

biotechnology 2018, 36 (8), 834-849. DOI: https://doi.org/10.1016/j.tibtech.2018.02.012.


Page 31 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4.


Pugh, C. W.; Ratcliffe, P. J., Regulation of angiogenesis by hypoxia: role of the HIF system.

Nature medicine 2003, 9 (6), 677. DOI: 10.1038/nm0603-677 5.

Bose, S.; Roy, M.; Bandyopadhyay, A., Recent advances in bone tissue engineering scaffolds.

Trends in biotechnology 2012, 30 (10), 546-554. DOI: 10.1016/j.tibtech.2012.07.005 6.

Vahabzadeh, S.; Bandyopadhyay, A.; Bose, S.; Mandal, R.; Nandi, S. K., IGF-loaded silicon

and zinc doped brushite cement: physico-mechanical characterization and in vivo osteogenesis evaluation. Integrative Biology 2015, 7 (12), 1561-1573. DOI: 10.1039/C5IB00114E 7.

Tamimi, F.; Sheikh, Z.; Barralet, J., Dicalcium phosphate cements: Brushite and monetite.

Acta Biomaterialia 2012, 8 (2), 474-487. DOI: https://doi.org/10.1016/j.actbio.2011.08.005. 8.

Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E., Matrix Elasticity Directs Stem Cell

Lineage Specification. Cell 2006, 126 (4), 677-689. DOI: https://doi.org/10.1016/j.cell.2006.06.044. 9. particle

Lammel, A. S.; Hu, X.; Park, S.-H.; Kaplan, D. L.; Scheibel, T. R., Controlling silk fibroin features

for

drug

delivery.

Biomaterials

2010,

31

(16),

4583-4591.

DOI:

10.1016/j.biomaterials.2010.02.024 10.

Konwarh, R.; Bhunia, B. K.; Mandal, B. B., Opportunities and challenges in exploring Indian

nonmulberry silk for biomedical applications. Proceedings of the Indian National Science Academy 2017, 83 (1). DOI: 10.16943/ptinsa/2017/41288 11.

Gupta, P.; Adhikary, M.; M, JC; Kumar, M.; Bhardwaj, N.; Mandal, B. B., Biomimetic,

osteoconductive non-mulberry silk fiber reinforced tricomposite scaffolds for bone tissue engineering. ACS applied materials & interfaces 2016, 8 (45), 30797-30810. DOI: 10.1021/acsami.6b11366 12.

Mandal, B. B.; Grinberg, A.; Gil, E. S.; Panilaitis, B.; Kaplan, D. L., High-strength silk protein

scaffolds for bone repair. Proceedings of the National Academy of Sciences 2012, 109 (20), 76997704. DOI: 10.1073/pnas.1119474109



13.

Page 32 of 45

Bhunia, B. K.; Kaplan, D. L.; Mandal, B. B., Silk-based multilayered angle-ply annulus

fibrosus construct to recapitulate form and function of the intervertebral disc. Proceedings of the National Academy of Sciences 2018, 115 (3), 477-482. DOI: 10.1073/pnas.1715912115 14.

Moses, J. C.; Nandi, S. K.; Mandal, B. B., Multifunctional Cell Instructive Silk‐Bioactive

Glass Composite Reinforced Scaffolds Toward Osteoinductive, Proangiogenic, and Resorbable Bone Grafts. Advanced healthcare materials 2018, 1701418. DOI: 10.1002/adhm.201701418 15.

Singh, Y. P.; Adhikary, M.; Bhardwaj, N.; Bhunia, B. K.; Mandal, B. B., Silk fiber

reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds. Biomedical Materials 2017, 12 (4), 045012. DOI: 10.1088/1748-605X/aa7697 16.

Singh, Y.; Moses, J. C.; Bhunia, B.; Nandi, S. K.; Mandal, B. B., Hierarchically Structured

Seamless Silk Scaffolds for Osteochondral Interface Tissue Engineering. Journal of Materials Chemistry B 2018. DOI: 10.1039/C8TB01344F 17.

Hutmacher, D. W., Scaffolds in tissue engineering bone and cartilage. In The Biomaterials:

Silver Jubilee Compendium, Elsevier: 2006; pp 175-189. DOI: 10.1016/B978-008045154-1.50021-6 18.

Guilak, F.; Cohen, D. M.; Estes, B. T.; Gimble, J. M.; Liedtke, W.; Chen, C. S., Control of

stem cell fate by physical interactions with the extracellular matrix. Cell stem cell 2009, 5 (1), 17-26. DOI: 10.1016/j.stem.2009.06.016 19.

Gupta, P.; Kumar, M.; Bhardwaj, N.; Kumar, J. P.; Krishnamurthy, C.; Nandi, S. K.; Mandal,

B. B., Mimicking form and function of native small diameter vascular conduits using mulberry and non-mulberry patterned silk films. ACS applied materials & interfaces 2016, 8 (25), 15874-15888. DOI: 10.1021/acsami.6b00783 20.

Koutsopoulos, S., Synthesis and characterization of hydroxyapatite crystals: a review study

on the analytical methods. Journal of Biomedical Materials Research: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 2002, 62 (4), 600-612. DOI: 10.1002/jbm.10280 32 ACS Paragon Plus Environment

Page 33 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21.


Gibson, I.; Rehman, I.; Best, S.; Bonfield, W., Characterization of the transformation from

calcium-deficient apatite to β-tricalcium phosphate. Journal of materials science: materials in medicine 2000, 11 (9), 533-539. DOI: 10.1023/A:1008961816208 22.

Hu, X.; Kaplan, D.; Cebe, P., Determining beta-sheet crystallinity in fibrous proteins by

thermal analysis and infrared spectroscopy. Macromolecules 2006, 39 (18), 6161-6170. DOI: 10.1021/ma0610109 23.

Magoshi, J.; Nakamura, S., Studies on physical properties and structure of silk. Glass

transition and crystallization of silk fibroin. Journal of Applied Polymer Science 1975, 19 (4), 10131015. DOI: 10.1002/app.1975.070190410 24.

Asakura, T.; Kuzuhara, A.; Tabeta, R.; Saito, H., Conformational characterization of Bombyx

mori silk fibroin in the solid state by high-frequency carbon-13 cross polarization-magic angle spinning NMR, x-ray diffraction, and infrared spectroscopy. Macromolecules 1985, 18 (10), 18411845. DOI: 10.1021/ma00152a009 25.

Enderle, R.; Götz-Neunhoeffer, F.; Göbbels, M.; Müller, F.; Greil, P., Influence of magnesium

doping on the phase transformation temperature of β-TCP ceramics examined by Rietveld refinement. Biomaterials 2005, 26 (17), 3379-3384. DOI: 10.1016/j.biomaterials.2004.09.017 26.

Wang, H.; Zhao, C.; Chen, Y.; Li, J.; Zhang, X., Electrochemical property and in vitro

degradation of DCPD–PCL composite coating on the biodegradable Mg–Zn alloy. Materials Letters 2012, 68, 435-438. DOI: https://doi.org/10.1016/j.matlet.2011.11.029. 27.

Liu, M.; Wu, C.; Jiao, Y.; Xiong, S.; Zhou, C., Chitosan–halloysite nanotubes nanocomposite

scaffolds for tissue engineering. Journal of Materials Chemistry B 2013, 1 (15), 2078-2089. DOI: 10.1039/C3TB20084A 28.

Pautke, C.; Schieker, M.; Tischer, T.; Kolk, A.; Neth, P.; Mutschler, W.; Milz, S.,

Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts. Anticancer research 2004, 24 (6), 3743-3748.



29.

Page 34 of 45

Hoppe, A.; Güldal, N. S.; Boccaccini, A. R., A review of the biological response to ionic

dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011, 32 (11), 27572774. DOI: 10.1016/j.biomaterials.2011.01.004 30.

Bandyopadhyay, A.; Bernard, S.; Xue, W.; Bose, S., Calcium Phosphate‐Based Resorbable

Ceramics: Influence of MgO, ZnO, and SiO2 Dopants. Journal of the American Ceramic Society 2006, 89 (9), 2675-2688. DOI: 10.1111/j.1551-2916.2006.01207.x 31.

Mirtchi, A. A.; Lemaitre, J.; Terao, N., Calcium phosphate cements: study of the β-tricalcium

phosphate—monocalcium phosphate system. Biomaterials 1989, 10 (7), 475-480.

DOI:

10.1016/0142-9612(89)90089-6 32.

Roy, M.; DeVoe, K.; Bandyopadhyay, A.; Bose, S., Mechanical property and in vitro

biocompatibility of brushite cement modified by polyethylene glycol. Materials Science and Engineering: C 2012, 32 (8), 2145-2152. DOI: 10.1016/j.msec.2012.05.020 33.

Alkhraisat, M. H.; Marino, F. T.; Retama, J. R.; Jerez, L. B.; Lopez-Cabarcos, E., Beta-

tricalcium phosphate release from brushite cement surface. J Biomed Mater Res A 2008, 84 (3), 710-7. DOI: 10.1002/jbm.a.31381 34.

Singh, Y. P.; Moses, J. C.; Bhardwaj, N.; Mandal, B. B., Injectable hydrogels: a new paradigm

for osteochondral tissue engineering. Journal of Materials Chemistry B 2018. DOI: 10.1039/C8TB01430B 35.

Knabe, C.; Adel‐Khattab, D.; Hübner, W. D.; Peters, F.; Knauf, T.; Peleska, B.; Barnewitz,

D.; Genzel, A.; Kusserow, R.; Sterzik, F., Effect of silicon‐doped calcium phosphate bone grafting materials on bone regeneration and osteogenic marker expression after implantation in the ovine scapula. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2018. DOI: 10.1002/jbm.b.34153 36.

Devi, K. B.; Tripathy, B.; Kumta, P. N.; Nandi, S. K.; Roy, M., In Vivo Biocompatibility of

Zinc-Doped Magnesium Silicate Bio-Ceramics. ACS Biomaterials Science & Engineering 2018, 4 (6), 2126-2133. DOI: 10.1021/acsbiomaterials.8b00297 34 ACS Paragon Plus Environment

Page 35 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

37.


Zhong, Z.; Qin, J.; Ma, J., Rapid synthesis of citrate-zinc substituted hydroxyapatite using the

ultrasonication-microwave method. Ceramics International 2017, 43 (16), 13308-13313. 38.

Midha, S.; Murab, S.; Ghosh, S., Osteogenic signaling on silk-based matrices. Biomaterials

2016, 97, 133-153. DOI: 10.1016/j.ceramint.2017.07.029 39.

Faia-Torres, A. B.; Guimond-Lischer, S.; Rottmar, M.; Charnley, M.; Goren, T.; Maniura-

Weber, K.; Spencer, N. D.; Reis, R. L.; Textor, M.; Neves, N. M., Differential regulation of osteogenic differentiation of stem cells on surface roughness gradients. Biomaterials 2014, 35 (33), 9023-9032. DOI: https://doi.org/10.1016/j.biomaterials.2014.07.015 40.

Fulmer, M. T.; Ison, I. C.; Hankermayer, C. R.; Constantz, B. R.; Ross, J., Measurements of

the solubilities and dissolution rates of several hydroxyapatites. Biomaterials 2002, 23 (3), 751-755. DOI: 10.1016/S0142-9612(01)00180-6 41.

Bose, S.; Tarafder, S., Calcium phosphate ceramic systems in growth factor and drug delivery

for bone tissue engineering: a review. Acta Biomaterialia 2012, 8 (4), 1401-1421. DOI: 10.1016/j.actbio.2011.11.017 42.

Sayer, M.; Stratilatov, A.; Reid, J.; Calderin, L.; Stott, M.; Yin, X.; MacKenzie, M.; Smith,

T.; Hendry, J.; Langstaff, S., Structure and composition of silicon-stabilized tricalcium phosphate. Biomaterials 2003, 24 (3), 369-382. DOI: 10.1016/S0142-9612(02)00327-7 43.

Bandyopadhyay, A.; Petersen, J.; Fielding, G.; Banerjee, S.; Bose, S., ZnO, SiO2, and SrO

doping in resorbable tricalcium phosphates: Influence on strength degradation, mechanical properties, and in vitro bone–cell material interactions. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2012, 100 (8), 2203-2212. DOI: 10.1002/jbm.b.32789 44.

Shih, Y. R. V.; Tseng, K. F.; Lai, H. Y.; Lin, C. H.; Lee, O. K., Matrix stiffness regulation of

integrin‐mediated mechanotransduction during osteogenic differentiation of human mesenchymal stem cells. Journal of Bone and Mineral Research 2011, 26 (4), 730-738. DOI: 10.1002/jbmr.278




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For Table of Contents Use Only

Synergistic effects of silicon/zinc doped brushite and silk scaffolding in augmenting the osteogenic and angiogenic potential of composite biomimetic bone grafts Joseph Christakiran Moses1, Mainak Dey 2, K. Bavya Devi 3, Mangal Roy 3, Samit Kumar Nandi 2, * and Biman B. Mandal 1, * 1Biomaterial

and Tissue Engineering Laboratory, Department of Biosciences and Bioengineering, Indian

Institute of Technology, Guwahati-781039, Assam, India 2Department

of Veterinary Surgery and Radiology, West Bengal University of Animal and Fishery Sciences,

Kolkata-700037, West Bengal, India 3Department

of Metallurgical and Materials Engineering, Indian Institute of Technology Kharagpur,

Kharagpur-721302, West Bengal, India *Corresponding

authors



Figure 1. A i) Scheme representing the methodology for fabricating (control) silk fibroin (SF) scaffolds and A ii) scheme representing the methodology for fabricating experimental silk doped brushite cement composite scaffolds; Elemental mapping reconstructed from the field emission electron micrographs for the different composite scaffolds; B) SF, C) BrC/SF, D) Si-BrC/SF, E) Zn-BrC/SF, F) Zn+Si-BrC/SF and G) pore size distribution analysed from electron micrographs.

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Figure 2. Compositional analyses ascertained through A) Fourier infrared spectra B) i) spectra showing blue shift in SF vs. BrC group deconvuluted Amide-I spectra for i) SF and ii) BrC/SF and C) X-ray diffractograms of different scaffolding groups used in the study; Uniaxial unconfined compressive mechanical studies on fabricated scaffolds; D) representative stress-strain curves for different scaffolds; E) Young’s modulus and F) compressive stress at 80% strain for different scaffolds used in the study; G) Unconfined cyclic mechanical assessment of the composite matrices; their compressive stress vs. time plot (# represents significant difference at p≤0.05 and $ represents significant difference at p≤0.01)



Figure 3. Osteogenic and osteoclastogenic potential of fabricated composite scaffolds; A) Live/dead cell imaging using calcein-AM (stains live cells green) and ethidium homodimer (stains dead cells red) for i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) MG63 cells on tissue culture plate (with insets of higher magnification); B) cellular viability using alamar blue assay; C) alkaline phosphatase activity assessment of the cell seeded constructs; D) tartrate resistant acid phosphatase (TRAP) staining for i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) osteoclasts differentiated on tissue culture plate (# represents significant difference at p≤0.05 and $ represents significant difference at p≤0.01); (multinucleated osteoclasts are represented with green arrows).


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Figure 4. Angiogenic potential of fabricated composite scaffolds; A) tube formation assay using calcein-AM stained porcine endothelial cells for conditioned media obtained from cell seeded constructs of i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF; vi) tube length and vii) number of tubes and nodes noticed per imaging field from the fluorescent micrographs; B) viability of seeded endothelial on silk matrices i) SF, ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, viii) percentage of dead cell population; C) proliferation of endothelial cells assessed by alamar blue assay and D) biochemical estimation of endothelial nitric oxide (NO) (# represents significant difference at p≤0.05)



Figure 5. In vivo evaluation of bone formation using histological examination of implanted scaffolds i) empty defect (control), ii) BrC/SF, iii) Si-BrC/SF, iv) Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) SF and fluorochrome labelling assessment vii) empty defect (control), viii) BrC/SF, ix) Si-BrC/SF, x) Zn-BrC/SF, xi) Zn+Si-BrC/SF, xii) SF at A) 1 month and B) 3 months post-implantation (Bright yellow region represents new bone (NB) regions whereas green regions represent old bone (OB) areas).


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Figure 6. Immunohistochemical assessment for expression of hypoxia inducible factor, HIF-1α (stained red) and bone sialoprotein, BSP (stained green) in different groups at 1 month, A) empty defect (control), B) BrC/SF, C) Si-BrC/SF, D) Zn-BrC/SF, E) Zn+Si-BrC/SF, F) SF, with nuclei counterstained with Hoechst 33342 (blue).



Figure 7. Radiographic 2D images of defect bone sites implanted with i) empty defect (control), ii) BrC/SF, iii) Si-BrC/SF, iv)Zn-BrC/SF, v) Zn+Si-BrC/SF, vi) SF; 3D Micro-CT images of defect bone sites implanted with vii) empty defect (control), viii) BrC/SF, ix) Si-BrC/SF, x) Zn-BrC/SF, xi) Zn+Si-BrC/SF, xii) SF showing extent of healing after A) 1month and B) 3 months (Red ring showing defect site; yellow arrow showing new bone regeneration)


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Table of Contents (TOC) Graphic 205x152mm (100 x 100 DPI)


zinc doped brushite and silk scaffolding in

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