Enzymatic Mineralization of Silk Scaffolds

Full Paper

Enzymatic Mineralization of Silk Scaffolds

Sangram K. Samal,* Mamoni Dash, Heidi A. Declercq, Tom Gheysens,Jolien Dendooven, Pascal Van Der Voort, Ria Cornelissen, Peter Dubruel,*David L. Kaplan*

The present study focuses on the alkaline pho
sphatase (ALP) mediated formation of apatiticminerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnatedwith different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatitewhile the structures differ as a function ofthe ALP concentration. Cellular adhesion,proliferation, and colonization of osteogenicMC3T3 cells improve on the mineralized SFPscaffolds. These findings suggest a simpleprocess to generate mineralized scaffoldsthat can be used to enhanced bone tissueengineering-related utility.
Dr. S. K. Samal, Dr. M. Dash, Prof. P. DubruelPolymer Chemistry and Biomaterials Research Group,Department of Organic Chemistry, Ghent University, Krijgslaan281/S4-Bis B-9000, Ghent, BelgiumE-mail: [email protected], [email protected]. S. K. Samal, Prof. D. L. KaplanDepartment of Biomedical Engineering, 4 Colby Street, TuftsUniversity, Medford, MA 02155, USAE-mail: [email protected]. H. A. Declercq, Prof. R. CornelissenDepartment of Basic Medical Science, Histology Group, GhentUniversity, De Pintelaan 185 (6B3) 9000, Ghent, BelgiumDr. T. GheysensAdvanced Concepts Team (ACT) European Space Agency, PPC-PF,Keplerlaan 1, PO Box 299 NL-2200, AZ, Noordwijk,The NetherlandsDr. J. DendoovenDepartment of Solid State Sciences, COCOON, Ghent University,Krijgslaan 281/S1 B-9000, Ghent, BelgiumProf. P. V. D. VoortDepartment of Inorganic Chemistry, COMOC, Ghent University,Krijgslaan 281 S3 9000, Ghent, Belgium

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1. Introduction
Biomineralization is the natural process bywhichminerals

are formed by organisms including proteins, peptides, and

enzymes.[1–6] Most of these mineralized products are

composite materials comprised of organic and mineral

components. Enzymatic-biomimeticmineralization involves

theuse of enzymes to control themicrostructureof inorganic

mineral apatite, which provides an important paradigm in

the design of biomineralized biomaterials. Alkaline phos-

phatase (ALP) is an importantenzymeinvolved intheprocess

of biomineralization.[7] ALP [phosphate-monoester phospho-

hydrolase (alkaline optimum); EC 3.1.3.1] is a homodimeric

enzymealsoknownasmetalloenzymeandeachcatalyticsite

contains three metal ions; two Zn and one Mg. The enzyme

is secreted by osteoblasts and ALP liberates phosphates

necessary for hydroxyapatite mineralization from organic

phosphates. The role of ALP has been extensively studied for

bone regeneration.

A promising biomaterial, silk fibroin protein (SFP), has

been extensively studied and also approved by the US Food

and Drug Administration for use in some biomedical

applications.[8–10] As a natural protein based biomaterial,

it has good mechanical properties, low inflammatory

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reactions, goodpermeability, andgoodbloodcompatibility.

The properties of silk are derived from its unique structure,

which consists of hydrophobic b-sheet crystalline domains

interrupted by hydrophilic amorphous acidic spacers.[11–13]

Recently, silk sponges and scaffolds have been used

extensively in bone tissue engineering applications and

facilitated bone formation in vitro and in vivo.[14–16]

Furthermore, silk fibroin regulates the formation of

hydroxyapatite when exposed to alternate soaking in

calcium- and phosphate-based solutions and in simulated

body fluid, which makes this protein an attractive

scaffolding material for osteoregenerative applications.[17]

Silk fibroin also controls the mineralization of calcium

carbonate, the inorganic phase of numerous invertebrate

species.[18] Moreover, silk has been shown to be a suitable

substrate for the adhesion of several cell types and

active growth of the cells on these silk substrates makes

it possible to combinemineral growthwith cell (osteoblast)

adhesion and differentiation.[19,20]

In the present study we expect, the acid (—COOH) and

amino (—NH2) groups of porous SFP scaffolds to chelate the

activemetallic sitesofALP (threemetal ions; twoZnandone

Mg). The lone pair electrons present on the nitrogen of

amino groups of SFP scaffolds can establish coordinate

bonds with metal ions of ALP. These interactions between

ALP and the active sites of SFP scaffolds lead to the

entrapment of the ALP within the scaffold. The porous

architecture of silk scaffolds further allows ALP to nucleate

and crystallize minerals and also provides a template for

vascularization and cell seeding. A possible reaction

mechanism is shown in Figure 1. As indicated by Golub

and Boesze-Battaglia in their review on the role of ALP, the

mechanismof inducingmineralizationbyALP involves two

possible mechanisms, a) increasing the local concentration

of inorganic phosphate, a mineralization promoter, and b)

decreasing the concentration of extracellular pyrophos-

phate, an inhibitor of mineral formation.[21] Thus, the

general concept is that ALP is involved in biomineralization

by cleavage of phosphate from organic phosphatemo-

noester such as b-glycerophosphate and catalyzes a trans-

phosphorylation reactionandassists in liberatinganexcess

of free inorganic phosphate species, a mineralization

promoter. This increased level of inorganic phosphate

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Figure 1. Proposed reaction mechanism of ALP with calcium phosph

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appears to be a crucial event in the initiation ofmineraliza-

tion in the presence of large concentrations of phosphate

acceptors.

In this study, we addressed the issue of improved

osteogenesis of biomaterial grafts by exploiting the

beneficial properties of silk scaffolds. The advantages of

using silk are numerous as silk-based grafts are not brittle

like most other calcium phosphate-based materials, and

these materials can exhibit mechanical properties close to

that of natural bone.[22,23] In addition, the silk scaffolds

consist of highly interconnected pores providing optimal

transport including oxygen and blood flow. Silk scaffolds

can also be mineralized and enable cells like osteoblasts to

adhere to the material. Enhanced mineralization of silk

scaffolds was studied as organic templates for induced

depositionof calciumphosphateusingALP. Ina secondstep,

thesemineralized interconnected porous silk scaffoldswere

seeded with osteoblasts to obtain bone-like tissue in vitro.ly

2. Experimental Section

2.1. Materials

Cocoons from Bombyx mori silkworm were obtained from Tajima

Shoji Co (Yokohama, Japan). Sodium carbonate (Na2CO3) and

lithium bromide (LiBr) were purchased as reagent grade from

Sigma–Aldrich or Fluka (St. Louis, MO) and used without further

purification. Dialysis cassettes (Slide-a-Lyzer MWCO 3.5K) were

purchased from Pierce biotechnology (Rockford, IL). Bovine

intestinal ALP (specific activity: �10 DEA units mg�1, P7640),

glycerol phosphate calciumsaltGP (50043), and calciumphosphate

powder (CaP) (230936) were purchased from Sigma (Sigma–

Aldrich, Belgium).

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2.2. Preparation of Aqueous Silk Fibroin Protein

Solution

Aqueous SFP solutions were prepared based on our published

protocols.[10,20] Briefly, whole cocoons were cut into small pieces

andwere boiled in a 0.02M aqueous solution of Na2CO3 for 20min.

The remaining fibroin was rinsed thoroughly in deionized water

and allowed to dry overnight. The dry fibroinwas thendissolved in

a 9.3M aqueous solution of LiBr at 60 8C for 6 h. The LiBr was

removed from the solution over the course of 48h by dialysis

ate.

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Table 1. Samples and their codes with respect to the amount ofALP soaked into the scaffolds.

Sample

ALP

[mg mL�1]

Sample

code

Salt leached SFP Scaffold – Silk-Sc

Scaffold mineralized without ALP 0 Silk-Bl

Scaffold mineralized with ALP 10 Silk-10




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cassettes (Slide-a-Lyzer MWCO 3.5K, Pierce Biotechnology, Rock-

ford, IL). The solution was centrifuged at 4 8C with 9000 rpm for

20min and filtered through a syringe based micro-filtration (5mm

pore size; Millipore Inc., Bedford, MA) to remove silk aggregates

formed during the process. This process enables the production of

8–10w/w% SFP in water. SFP solutions with lower concentrations

were prepared by diluting the above solutionwith double distilled

deionized water. The final SFP concentration of the solution was

monitored by drying 1ml silk solution samples in a plastic petri

dish at 60 8C (American Scientific Products, Constant Temperature

Oven,Model DK-42) andweighing the resulting dried films. All the

sample experiments were performed in triplicate.


2.3. Silk Fibroin Protein Scaffold Fabrication

Salt leached porous SFP scaffolds were prepared according to the

procedure described in literature.[24,25] Briefly, 4 g of the granular

NaCl was added slowly to a cylindrically shaped container with

2ml of SFP solution. The container was covered to reduce

evaporation rate and kept at room temperature for homogeneous

distributionof thesolution.After24h, thecontainerwasuncovered

and immersed in MilliQ water with stirring for 24h to leach out

NaCl particles. The desired dimension of the SFP scaffold was

punch-out with punch-pressure equipment.

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2.4. ALP Quantification in SFP Scaffold
ALP activity was measured by monitoring the color change of p-nitrophenol phosphate (pNPP) (colorless) into p-nitrophenol(yellow). Different concentrations of 20mL ALP were impregnated

into two sets of SFP scaffolds and kept for 2 h for complete

absorption. One set of ALP absorbed SFP scaffolds was used to

determine the amount of ALP absorbed inside the SFP scaffold. The

ALP absorbed scaffolds were housed in Eppendorf tubes. One

milliliter of MilliQ H2O was added and sonicated for 3� 10 s

(amplitude of 40%) (Vibra Cell SONICS (ANALIS)) to observe the

amount of ALP physically absorbed. After sonication, the superna-

tant was used for ALP quantification. One microliter of the

supernatant was added to 50mL of water and 50mL p-nitrophenylphosphate (4.34mM in 100mM glycine, pH 10.3, 1mM MgCl2 in

MilliQwater)wasadded to themixtureand incubated for 15minat

room temperature on a bench shaker. The enzymatic reaction was

stoppedbyadding50mLof1MNaOH.Enzymeactivityasquantified

by absorbance measurements at 405nm (Universal Microplate

Reader EL800, BIO-TEK instruments) and calculated according to a

series of ALP standards. The other set of ALP absorbed SFP scaffolds

was used for biomineralization and after 3 d of mineralization the

ALP content of the SFP scaffoldswas determined as describe above.

Each set of samples were analyzed in triplicate.

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2.5. ALP Mediated Scaffold Mineralization

Thescaffoldswere impregnatedusingsolutionof fourdifferentALP

concentrations; 10, 20, 40, and 60mgmL. The sample codes with

respect to the ALP concentration used for the biomineralization

process are shown in Table 1. The porous silk scaffolds with

dimension 5mm�5mm were allowed to impregnated slowly

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500mL of different concentration of ALP into the pores of the

scaffold. The ALP solution containing scaffolds were allowed to

stand in air for 2 h to allow the ALP to get well absorbed into the

pores of the scaffold such that theALPwas notwashed awaywhen

placed in the 0.1M GP medium. The swollen semi-dried scaffolds

were placed into themineralizationmedium consisting of 0.1M GP

(aq)mediumand induce theonsetof thebiomineralizationprocess.

The mineralization medium was changed every day. After 7 d of

mineralization, gels were rinsed three times in Milli-Q water to

remove residualGPand subjected to lyophilization after freezingat

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2.6. Mass Increase of Mineralized SFP Scaffold

The mass increases of the mineralized scaffolds were determined

byweighedof freeze-driedmineralized scaffold (after incubation in

0.1MGP for 7dand freezedried) in triplicate. Themass increasewas

calculated using the following equation:

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Mass increase ð%Þ ¼ ðMf �MiÞMi

� 100

whereMf is the final mass of scaffolds after freezedrying.Mi is the

initial mass of scaffolds before incubation and mineralization

treatments. The final mass increase was calculated by using the

same formula as above but with mt being the dry mass of the

scaffolds after mineralization.

2.7. Scaffold Morphology by Scanning Electron

Microscopy (SEM) and Composition by Energy

Dispersive X-Ray Spectroscopy (EDX)

SEManalysiswas performedon a FEI Quanta 200F instrument (FEI,

Eindhoven, The Netherlands) in the secondary electron mode. The

microscope is linked toanEDXsystem(EDAXGenesis 4000, Tilburg,The Netherlands) for elemental analysis. Prior to analysis, all

sampleswere freeze-dried for 48h. The scaffoldswere fractured by

using tweezers followedby coatingwith a thin conductive layer. In

the case of SEM-EDX analysis, samples were coated with a thin

carbon layer by flash evaporation. In case of SEM examination,

samples were coated with a thin gold layer using an Emitech

SC7620 Sputter Coater (Quorum Technologies, Kent, UK).

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2.8. Mechanical Analysis of Mineralized SFP

Scaffolds

Compression tests on cylindrical SFP scaffolds samples of 10mmin

diameter and 6mm height were performed using an Instron

Machine (HT, HOUNSFIELD, Eke, Belgium) equipped with a 0.1 kN

load cell at 24 8C. An initial compressive contact to 0.05N was

applied to ensure complete contact between the sample and the

surface. Load and displacement data were collected during the

experiments at a constant crosshead speed of 1mmmin�1 and

until 60%deformation in specimenheight. Compressive stresswas

presented by its nominal value s, which is the force per cross-

sectional area, while the strain is given by the deformation ratio a,

the deformed length per initial length of the specimen. After the

compression tests, the compressive stress and strainwere graphed.

The compression elastic modulus was calculated from the slope of

the initial linear domain of the stress–strain curves between 0 and

5% compressions. At least, five samples were measured for each

mineralized samples and the average value results are reported.

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2.9. Attenuated Total Reflectance-Fourier Transform

Infrared (ATR-FTIR) Spectroscopy Analysis

ATR-FTIR measurements were carried out using a Biorad FT-IR

spectrometer FTS575C (Bio-Rad, Nazareth, Belgium) equippedwith

a ‘‘GoldenGate’’ ATRaccessory (Specac,Kent,UnitedKingdom). The

latter was fittedwith a diamond crystal. The spectrawere taken as

an average of 32 scans with 4 cm�1 of resolution in the region of

5000–400 cm�1 using a WIN-IR software.

2.10. Fourier Transform-Raman (FT-Raman)

Spectroscopy

FT-Raman spectrawere performedonanFT-RamanModule (NXR,

Thermo Fisher Scientific,Madison,WI, USA). The powder samples

were pressed into a gold-coated sample holder and spectra

were collected with a laser power of 0.35W and number of

scans of 1500 at a resolution of 4 cm�1. The spectrawere obtained

in the Raman shift region between 400 and 4000 cm�1 using

OMNIC software (Version 5.1, Thermo Fisher Scientific, Madison,

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2.11. Thermogravimetric Analysis (TGA)

TGA experimentswere performedwith a TA Instruments, TGAQ50

thermo-balance (TA Instruments, Ghent, Belgium) with Thermog-

ravimetricAnalyzer software (UniversalAnalysis, 2000). The freeze-

dried mineralized scaffold sample weights were between 4 and

6mg andwere scanned at 10 8Cmin�1. The temperature rangewas

30 to 700 8C under a 60mLmin�1 flow rate of nitrogen.[26]

2.12. X-Ray Diffraction (XRD)

XRD was used to characterize the phase of the formed calcium

phosphate mineral deposited in the silk scaffolds. The diffraction

patterns were collected using a Siemens Kristalloflex D5000




diffractometer (Siemens, Karlsruhe, Germany) working in the

Bragg–Brentano configuration and equipped with a Cu Ka X-ray

tube, a diffracted beam monochromator and a scintillation

detector. The samples were scanned over a 2u range of 5–608with

a step size of 0.028 and a time per step of 2 s.

2.13. Cell Culture Studies

2.13.1. Cell Culture and Cell Seeding onto Scaffolds

MC3T3-E1 cells (mouse calvaria preosteoblast cells, sublcone 14,

ATCC) were cultured in a-MEM L-glutamax (Gibco Invitrogen)

supplemented with 10% fetal calf serum (FCS, Gibco Invitrogen)

and 0.5 vol% penicillin–streptomycin (10 000UmL�1–10 000mg

mL�1, Gibco Invitrogen) (standardmedium). Cells were cultured at

37 8C in a humidified atmosphere of 5% CO2. The silk scaffolds,

blank silk scaffolds andmineralized silk scaffolds (d 5mm,h5mm)

were sterilized using ethylene oxide cold cycle (Maria Middelares,

Ghent, Belgium). Before cell seeding, the scaffolds were immersed

in serum-free a-MEM medium in 24-well plates. After 24h, the

scaffolds were placed into 24-well tissue culture dishes (for

suspension culture). Cells were seeded at a density of 333 000

cells/40mL/scaffold for cell viability/proliferation and 1.106 cells

for colonization, and were allowed to adhere for 4h. Standard

culture medium (160mL) was added to each well and the seeded

scaffoldswere further incubatedovernight. After 24h, cell/scaffold

constructs were placed in 12-well plates. Standard medium (3mL)

was added and the cell/scaffold constructs were cultured for 21 d

(5% CO2/95% air, 37 8C).

2.13.2. Characterization of Cell/Scaffold Constructs

Cell adhesion, proliferation, and colonization were evaluated at

different time points with the following analyses.

2.13.2.1. Fluorescence microscopy

To visualize cell adhesion and colonization of the scaffolds, cell/

scaffold constructs were evaluated using fluorescencemicroscopy

after performing live/dead staining. After rinsing with PBS, the

supernatant was replaced by 1mL PBS solution supplemented

with 2mL (1mgmL�1) calcein AM (Anaspec, USA) and 2mL

propidium iodide (1mgmL�1) (Sigma–Aldrich). Cultures were

incubated for 10min at room temperature,washed twicewith PBS

solution and evaluated by fluorescence microscopy (Olympus

Inverted Research SystemMicroscope, type U-RFL-T, Cell software,

Olympus, Belgium). Evaluations were done post-seeding at Day 1,

7, and 14.

2.13.2.2. Prestoblue viability

The Prestoblue assay (Invitrogen) was applied to quantify cell

viability and proliferation in the cell/scaffold constructs. Pres-

toblue is a blue non-fluorescent, cell permeable compound

(resazurin-based solution), that is reduced by living cells into a

fluorescent compound (resorufin). 100mL PrestoBlue reagent was

added to 900mL culture medium/scaffold and incubated for 2 h at

37 8C.Thefluorescence intensitywasperformedontheWallac1420

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Viktor 3TM plate reader (PerkinElmer, Inc.) at 535nm excitation

and 615nm emission. Triplicate measurements were performed

post-seeding at Day 1, 7, 14, and 21.

2.13.2.3. Histology

Cell/scaffold constructs were rinsed with Ringer solution, fixed

with 4% phosphate (10mM) buffered formaldehyde (pH 6.9) (4 8C,24h), dehydrated in a graded alcohol series and embedded in

paraffin. The scaffolds were sectioned 5–7mm and stained with

hematoxylin& eosin (H&E) andmountedwithmountingmedium

(Cat.No. 4111E, Richard-Allan Scientific).

3. Results

3.1. Salt-Leaching Silk Scaffold Fabrication

The NaCl salt-leaching scaffold fabrication protocol is an

easy, effective, andwidelyused fabricationprocess in tissue

engineering applications.[27,28] The scaffolds prepared by

salt leaching method were porous with interconnected

pores due to salt templating, enabling good access for the

medium. The salt-leached silk scaffolds generally are stiffer

and possess rough surface due to the partial solubilization

of NaCl and b-sheet formation, which improved cell

attachment and proliferation.[24,29]

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3.2. ALP Quantification in SFP Scaffold
ALP absorption into the SFP scaffolds after 2 h of incubation

is shown in Figure 2. The SFP scaffolds chelated ALP via the

carboxylic and amine groups to around�50% of ALP, while

the remaining ALP was in the supernatant after sonication

and washing. After 3 d of mineralization the ALP

quantification results indicated �10% of the remaining

ALP leached out from the mineralized scaffolds (Figure 2).r Pers

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Figure 2. ALP content of SFP scaffold after 2 h ALP absorptionand 3 d mineralization.




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3.3. Scaffold Biomineralization and Mass Increase

TheALP incorporated into silk protein scaffolds followed by

incubation in a solution containing 0.1M GP and mineral-

ized for one week. An indirect indicator of mineralization

is the mass gains observed during the mineralization

treatment are shown in Figure 3. In allmineralized samples

weight increase of more than 150% was observed due to

mineral deposition.

3.4. Mineral Morphological and Compositions

Analysis by SEM and EDX Analysis

The microstructures of silk scaffolds produced by freeze-

drying are shown in the SEMs images in Figure 4. The cross-

sectional morphology of the scaffolds was obtained by

fracturing the scaffolds with tweezers along their cross-

sections and followed by metal sputtering process. An

interconnected porous microstructure was observed in the

cross-section of the scaffolds. The scaffolds mineralized for

one week in the presence of different ALP concentrations

exhibitedmineral deposits on thewalls of silk scaffolds. The

mineral crystals were dense and homogeneously covered

the surfaceof thepores.Moreover, the crystalmorphologies

differed as a function of the applied different ALP

concentration. This was further confirmed at higher

magnification, where clusters of crystals with different

shapes could be distinguished for the different ALP

concentrations applied. In the scaffold with lower ALP

concentration, that is, Silk-10, the crystals were present as

rounded clusters. Upon increasing the enzyme concentra-

tion, themorphologyof themineral clusters changed. In the

Silk-40 and Silk-60 scaffolds, the crystals were present in

the formof dendrites. The EDXanalysiswas then applied to

determine the different Ca/P fraction in different mineral-

ized scaffolds. The presence of calcium, phosphorous, and

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Figure 3. Mass change after 7 d of blank scaffold without enzyme(Silk-Bl) as control and ALP-mediated mineralized silk scaffolds(Silk-10, Silk-20, Silk-40, and Silk-60) in triplicate with standarddeviation.

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Figure 4. SEM-EDX images of the salt leached silk scaffold (Silk-Sc), blank scaffold without enzyme (Silk-Bl) as control and differentconcentration ALP-mediated mineralized scaffolds Silk-10 (ALP 10mgmL�1), Silk-20 (ALP 20mgmL�1), Silk-40 (ALP 40mgmL�1), Silk-60 (ALP60mgmL�1) silk scaffolds.

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oxygen indicates the possible presence of calcium phos-

phate in the scaffolds.

3.5. Mechanical Analysis of Mineralized SFP

Scaffolds

The mechanical analysis showed that ALP mineralized

scaffolds at all concentrations had higher compressive

strength compared to the blank (non-mineralized) control




scaffolds (Figure 5).However, surprisingly the scaffolds Silk-

10 and Silk-20 had a higher compressive strength than the

scaffolds at higher ALP concentrations (Silk-40 and Silk-60).

3.6. Evidence of Mineral Deposition by ATR-FTIR

TheATR-FTIR is an interestinganalysis technique to confirm

thepresenceof thefunctionalgroupsof theformedminerals

in the mineralized SFP scaffolds. The general characteristic

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Figure 5. Mechanical analysis of mineralized SFP scaffold a) compressive stress–strain curve and b) compressive modulus.


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vibration peak of the amide groups in SFP appears

between 1600 and 1700 cm�1 for amide I (C55O stretching),

between 1550 cm�1 and 1500 cm�1 for amide II (NH

bending), and between 1270 and 1230 cm�1 for amide III

(C—N and N—H functionalities). In general, amide I region

1650–1660 cm�1 associate with a-helixs conformation,

whereas 1640–1650 cm�1 represents the random coil

conformation and peak bands 1620–1640 cm�1 for b-sheet

conformation.

A clear difference was observed in the ATR-FTIR spectra

(Figure 6) of themineralized scaffolds with that of the non-

mineralized scaffolds. The broad band appearing in the

region 3300–3500 cm�1 was assigned to the vibrational

band of the OH-groups and in general can be assigned to

moisture present in the samples. Figure 6 shows amide I

peak at 1620 cm�1, amide II adsorption at 1512 cm�1, and

amide III peak at 1231 cm�1 for SFP scaffold. In the case of

the mineralized scaffold, amide I and amide II absorption

happens at the same wave numbers as that of non-

mineralizedSFP scaffold.However, thepeak intensityof the

mineralized scaffold is less due to the presence of minerals

in themineralizedscaffold. The intensityof theamidepeaks

of the mineralized scaffold is less compared to that of the

silk-scaffold. The mineralized SFP scaffolds shows typical

peaks seen at 1050–1060 cm�1 and 980–999 cm�1 (P—OFor

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Figure 6. FTIR-ATR spectra of the salt leached silk scaffold (Silk-Sc), blanmineralized scaffolds (Silk-10, Silk-20, Silk-40, and Silk-60) silk scaffoscanned over the range 1700–500 cm�1.




stretching), and 655–660 cm�1 (O—P—O bending). The

band at 980–999 cm�1 reflects the n1 band of the P—O

stretching mode, which became sharper and more promi-

nent with increasing enzyme concentration. The weak

bands at 870-872 cm�1 (C—O stretching) are derived from

carbonate ions.

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3.7. Raman spectroscopy

In this study Raman spectroscopy was applied to study

the structural properties of SFP scaffolds, before and after

mineralization (Fig. 7). The silk scaffolds displayed charac-

teristicbandsat1664,1230,1080cm�1,whichareattributed

to b-sheets.[30] There are also relatively weak bands present

at1261cm�1associatedwitha-helices.Ramanspectroscopy

revealed a strong peak at 960 cm�1 for the enzymatically

mineralized silk scaffolds, which was absent in both native

silk scaffolds and scaffolds without enzyme.

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3.8. Thermogravimetric Analysis (TGA)

The thermal stability of the mineralized scaffold and

quantification of the amount of mineral deposit on the silk

scaffolds were analyzed by TGA. The TGA traces of the silk

k scaffoldwithout enzyme (Silk-Bl), CaP as control and ALP-mediatedlds a) scanned in the range 4000–500 cm�1 and b) enlarged view,

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Figure 7. FT-Raman spectra of the salt leached silk scaffold (Silk-Sc), blank scaffold without enzyme (Silk-Bl), CaP as control and ALP-mediated mineralized scaffolds (Silk-10, Silk-20, Silk-40, and Silk-60) silk scaffolds a) scanned in the range 250–3500 cm�1 and b) enlargedview, scanned over the range 600–1700 cm�1.

Figure 8. a) TGA spectra and b) residue at 700 8C of the salt leached silk scaffold (Silk-Sc), blank scaffold without enzyme (Silk-Bl) as a controland mineralized silk scaffolds (Silk-10, Silk-20, Silk-40, and Silk-60).

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scaffolds as well as the mineralized scaffolds are shown in

Figure8a.All the traces showacontinuousweight lossup to

700 8Cwith a first step loss in the temperature range of 30–

150 8C attributed to the loss of moisture, followed by

continuous weight loss from 250 8C onwards due to the

polymer decomposition at elevated temperatures. The

thermal investigation by TGA led to the observation that

mineralized scaffolds (containing ALP) possessed higher

residue content at 700 8C (Figure 8b) due to the presence of

minerals compared to non-mineralized scaffolds. The first

degradation step in the temperature range of 30–150 8Ccorresponded to the equilibrium moisture of the samples.

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Figure 9. X-ray diffraction pattern of the salt leached silk scaffold(Silk-Sc), blank scaffold without enzyme (Silk-Bl) as control andmineralized silk scaffolds (Silk-10, Silk-20, Silk-40, and Silk-60).

3.9. XRD Analysis

The X-ray diffractograms of the scaffolds mineralized for

different timeperiodsareshowninFigure9.All thepatterns

showed a strong reflection with a broad peak around 318(2u), showing that the mineral formed on the silk scaffolds

was apatite. The peak was broader and not well resolved,

with a noise level in the diffraction data due to the

amorphous properties of the biopolymer in the scaffold.




3.10. Cell Culture Studies

3.10.1. Cell Viability, Adhesion, and Proliferation

Cells on the scaffolds were examined by fluorescence

microscopy after live/dead staining (Figure 10) and the

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Figure 10. Cell viability and proliferation on control and mineralized silk scaffolds. Fluorescence microscopy (CaAM/PI staining) of MC3T3cells cultured on the scaffolds for a–e) 1 d, f–j) 7 d, and k–o) 14 d, for a,f,k) the Silk-Bl control scaffolds andmineralized scaffolds, b,g,l) Silk-10,c,h,m) Silk-20, d,i,n) Silk-40, and e,j,o) Silk-60.


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viability was followed for 21 d with the Presto Blue

(Figure 11). One day after seeding (Figure 10 1D), MC3T3

cells were viable (stained green) and the amount of cells

on each of the mineralized scaffold appeared similar

and fewer cells were detected on the control silk scaffolds.

The amount of dead cells (stained red) was limited on all

the scaffolds. It should be noted that the silk scaffolds

demonstrated high background fluorescence after PI

staining. The cells on the scaffolds, independent of the

mineralization status, were well spread and showed a




polygonal morphology (Figure 10). These qualitative data

were confirmed by Presto Blue assay, after 1 d, a higher

number of cells attached to mineralized silk scaffolds

(Silk-10 to Silk-60). After 7–14 d, the surface of the control

silk scaffold and mineralized scaffolds were covered with

viable, well-attached cells (Figure 10f–o). After 7, 14, and

21 d, the highest number of cells was detected on the

Silk-10 and Silk-20 scaffolds (Figure 11). An increase in

mineral content (like Silk-20 to Silk-60) resulted in

decreased cell viability.

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Figure 11. Viable cells cultured on Silk-Bl (control) andmineralized(Silk-10, Silk-20, Silk-40, and Silk-60) silk scaffolds for over 21 d.The amount of viable cells was quantified with the Prestoblueassay and represented as relative fluorescence units (RFU).

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3.10.2. Cellular Colonization of the Scaffolds

Cross-sections of paraffin-embedded cell/scaffold con-

structs after 7, 14, and 21 d in culture are presented in

Figure 12. Cell layers were predominantly formed at the

edges of the scaffolds, clearly following the contours of the

scaffolds. The quantitative data in Figure 11 further reveal

decreased cell viability levels as a function of increasing

enzyme concentrations. on
4. Discussion
Bone is a composite of biological polymer and calcium

phosphate mineral possessing interconnected macro- and

micro-porous structures. Commonly available commercial

bone graft substitutes are based on calcium phosphate

ceramics, titanium, or bioactive glasses. The currently

available bone substitute biomaterials often fall short to

fulfill thedesired requirements forbone tissue regeneration

including the durability, the mechanical properties with

surrounding tissue, and an appropriate rate of resorption.

The scaffolds for bone tissue formation require suitable

porosity to facilitate cell attachment, proliferation, migra-

tion, nutrient, and waste transport into and out of the

scaffold system.[27,29]

A number of fabrication methods have been utilized to

design porous interconnected silk-based scaffolds. Porous

silk sponges can be fabricated using freeze-drying, gas

foaming, among other methods.[13,31,32] Among these,

NaCl salt-leaching is an easy, effective, and widely used

process.[27,28] The scaffolds prepared by salt leaching had

interconnected pores due to salt templating, enabling good

access for the medium. In this process, the surface of the

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NaCl particles dissolves in the SFP aqueous solution, while

most of the NaCl is retained in the solid state because of

super saturation of the solution. The primary sequence of

the SFP contains the heavy chain dominated by hydropho-

bic domains, with two large hydrophilic blocks at ends of

the chain.[33] The hydrophobic domains in SFP constitute

around 79% of the total amino acids, with GAGAGS peptide

repetitive sequences dominating b-sheet structures that

form the crystalline regions in SFP based biomaterials.[34,35]

In salt leaching,proteinsolubilitydecreaseswith increasing

salt concentration, the interactions between different

domains (heavy chains, hydrophobic, and hydrophilic

chains) become favored from both intra- and interchain

interactions. The non-polar residue interactions are more

pronouncedwith the addition of salt, leading to the salting-

out effect.[36] Thebehavior of thefibroin in the concentrated

NaCl systemmaybe related to the role of the salt ions in the

extracting medium that would otherwise coat the hydro-

phobic fibroin domains, promoting chain–chain interac-

tions leading to a newmore stable structure. These changes

in hydrophobic hydration induce protein folding, resulting

in b-sheet formation.[37] After 24h, the SFP aqueous

solutions convert to a water stable interconnected porous

scaffold. The salt-leached silk scaffolds are generally stiffer

and possess rough surfaces due to the partial solubilization

of NaCl and b-sheet formation, which improves cell

attachment and proliferation.[24,29]

Silk protein scaffolds have been applied as protein-based

biomaterial systems for tissue engineering.[16,38] The

shortcoming of silk scaffolds for bone tissue engineering

applications include the lack of osteogenic features and

the need to improve mechanical properties relative to

native bone. There have been several strategies to

incorporate calcium phosphate with porous silk fibroin

polymeric scaffolds to generate organic/inorganic compo-

sites.[14,15] These inorganic particles act as nucleation sites,

which enable further mineralization. Each of these strate-

gies to incorporate calcium phosphate have advantages

and disadvantages, including the aggregation of inorganic

particles leading to uneven dispersion and poor reproduc-

ibility, inadequate size, shape, and complicated processing

steps. In the present study, we focused on a simple

enzymatic mineralization strategy to introduce calcium

phosphate biomineral into SFP scaffolds. The ALP is a

homodimeric enzyme, also known as metalloenzyme and

each catalytic site of ALP consist of threemetal ions, that is,

two Zn and one Mg, necessary for enzymatic activity. The

porous SFP scaffolds comprises of amino acids and free

amino groups, which allow to chelate to the active metal

sites of ALP. The lone pair electrons present on the nitrogen

of amino group can establish coordinate bonds with metal

ions. Thedifferentaminoacidsandaminogroupspresentat

the nucleation process influence the morphologies and

types of crystals. The porous architecture of the silk scaffold

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Figure 12. Colonization of MC3T3 cells on control and mineralized silk scaffolds. Histological analysis (H&E staining) of MC3T3 cells culturedon 3D scaffolds for 7, 14, and 21 d. a) Silk-Bl (control) and b) mineralized Silk-10, c) Silk-20, d) Silk-40, and e) Silk-60.


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allows ALP to nucleate and crystallize minerals and also

provides template for vascularization and cell seeding. The

establishment of coordinate bond during chelation allows

ALP to remain entrappedwithin the porous structure of the

SFP scaffolds and initiate nucleation in a calcium glycer-

ophosphate medium. The amount of ALP absorbed in SFP

scaffolds was determined and results indicate that the ALP

was encapsulated by chelation and physical entrapment.

TheALPcontainingSFPscaffolds followed incubation for7d




in a solution containing 0.1M GP. The calcium ions and

glycerophosphate diffused into the silk scaffolds contain-

ing ALP. The ALP cleaves phosphate ions from glycerophos-

phate,whichare then free to reactwithcalciumions to form

insoluble CaP, which precipitated and remained trapped

within the scaffold, while by-products such as glycerol are

free to diffuse out of the material. An indirect indicator

of mineralization is the mass gain observed during the

mineralization process.

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S. K. Samal et al.

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The scaffolds treated with increasing concentrations of

ALP showed increased dry weight compared to the non-

mineralized scaffolds. The different concentrations of ALP

form different structures and sizes of mineral phases.

Increasing ALP concentration resulted in mineralized SFP

scaffolds thatwerebrittle and someof themineral particles

were also extracted in water. The data for the mineralized

scaffolds shows mass increase related to the addition of

combination of organic and inorganic components. The

lowest ALP concentration provides the right environment

to initiate some precursor (organic components) prior to

inorganicminerals formation. Thehigher concentrations of

ALP are sufficient enough to prepare inorganic minerals

with less organic components. Other interpretationsmight

be thatupontheenzymeabsorptionwhenthesilk surface is

saturated with enzyme, further deposition only occludes

the underlying enzyme, thus different structure are

observed but no further increase in mineral activity is

realized. The different enzyme concentrations did not

show a trend in scaffold mass increase due to the presence

of different minerals, which have different structural and

compositional properties, and was further confirmed by

SEM and EDX analysis.

An interconnected porous microstructure was observed

by SEM for the salt leached scaffolds. The mineralized

scaffolds exhibited mineral deposits on the silk walls. The

mineral crystals were dense and homogeneously covered

the surfaces of the pores. Moreover, the crystal morpho-

logies differed as a function of the applied different ALP

concentration. Thisdifference inshapescanbeattributed to

the fact that different types of mineral precursors were

formed upon exposure to different concentrations of ALP.

The EDX analysis confirms the presence of calcium,

phosphorous, and oxygen, which indicates the possible

presence of calcium phosphate in the SFP scaffolds. The

mechanical properties of these scaffolds revealed that

the compressive modulus of mineralized scaffold did not

directly correlate with increased ALP concentration, possi-

bly due to the formation of different types and sizes of

calcium phosphate mineral precursors. Other interpreta-

tionsmight be that upon the enzyme adsorption, when the

silk surface is saturated with enzyme, further deposition

only occludes the underlying enzyme, thus, no further

increase in activity is realized.

ATR-FTIR shows the intensity of the amide peaks of the

mineralized scaffold is less compared to that of the silk-

scaffold, which arises due to the formation of the bond

between the Ca2þ ions and C55O. The oxygen atoms of the

carboxyl and carbonyl groups present on the surface of the

SFP scaffold bind with the calcium ions, and they serve as

the nucleation sites for mineral formation. Subsequently,

the minerals precipitate on the surface of the SFP scaffolds.

The weak bands at 870–872 cm�1 (C—O stretching) are

derived from carbonate ions,which indicate that PO3�4 sites

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in the synthesized apatite calcium phosphate are replaced

partiallyby carbonate ions and thisband is absent in caseof

blank scaffolds that are non-mineralized. The CO2�3 is

incorporated into the scaffold duringmineral precipitation.

Additional analysis by Raman and XRD confirmed the

formation of calcium phosphate and carbonate in the

scaffolds. These data together with the EDX, further

suggests thepresenceof calciumphosphate in thescaffolds.

The highest concentration of the ALP lead to the most

heavily mineralized scaffolds, thus the enzyme mediated

the mineralization by freeing phosphate from the GP. The

mineralized scaffolds had a calcium phosphate mineral

content. The proposed enzymatic mineralization with ALP

is an easy, efficient, and controllable way of mineralizing

silk scaffolds.

Thermal analysis results indicated that theweight losses

of the mineralized scaffolds was small compared to that of

the non-mineralized scaffolds, indicating the presence of

a larger amount of inorganic mineral in the scaffolds. The

residualweight of Silk-B1 is due to someglycerol phosphate

that is still remaining inside the scaffold after washing.

The residue in Silk-Sc and also in Silk-B1 arises due to the

nitrogen environment used during TGA analysis. The non

volatile residues remain due to the formation of highly

condensed carbon char or aromatic structures and ash

under nitrogen atmosphere. TGA curves of higher ALP

concentrations were similar to the lower ALP-containing

scaffolds, even if therewas a difference in themass change.

Since, the lowest ALP concentration provided a suitable

environment to initiate precursor (organic components)

composition prior to inorganic mineral formation. The

higher concentrations of ALP were sufficient to prepare

inorganic minerals with less organic precursor compo-

nents. Another interpretation might be that upon enzyme

absorption the silk surface may become saturated with

enzyme, thus further deposition occluded the underlying

enzyme, thus different structures were observed but no

further increase in mineral activity was realized. TGA

analysis only estimates the inorganic minerals present in

the samples, which is the reason that TGA curves of higher

ALP concentrations were similar to the lower ALP contain-

ing scaffolds, even with a difference in the mass change.

By seeding the mineralized scaffolds with cells, it was

noted that the cells were evenly distributed and similar for

the differentmineral contents. Cell proliferation correlated

inversely with silk mineralization with 20% ALP after 21 d.

Cell proliferation with scaffolds with 60% ALP was not

significantly different after 21 d. ALP40 and ALP60

supported fewer cells than the ALP20 and this could be

due to a number of factors. For example, an increase in

mineralization close the interconnectivity of the pores

leading to a decreased integration of cells into the scaffold,

the calciumphosphate crystals areneedle shapedandcould

be detrimental to the cells, the cells grow on small sized

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apatite and do not adhere properly and being removed

during media changes.

Initially, it was thought that the scaffolds with the

highest degree of mineralization would be the best for cell

responses, however the reverse was noted. There seems to

be a trade-off between the amount of mineralization and

cell responses. In vivo calcium phosphate is converted to

hydroxyapatite where the calcium phosphate crystals act

as nucleation sites. Thicker mineralization would slow this

conversion, implying that lower concentrations would be

better for bone-related goals for the 20% ALP scaffolds.

These systems could be utilized as post mineralized

scaffolds with or without osteogenic cells for bone tissue

engineering applications.

5. Conclusion

The present study demonstrates a simple and efficient

enzyme-mediated apatite deposition strategy to fabricate

SFP biomineralized scaffolds. The porous SFP scaffoldswere

homogeneously mineralized at physiological conditions.

ATR-FTIR, Raman, and XRD analyses confirmed the

structure of theminerals as calcium phosphate. The results

indicate that 20mgmL�1 ALPmineralized silk scaffolds can

maximize osteoblast differentiation for bone tissue engi-

neering applications.

The authors declare no competing financial interest.
n Acknowledgements: The authors would like to thank XiaoqinWang, Carmen Preda, Tom Planckaert, and Timothy Douglas fortheir help. The authors would also like to thank EP2CON(IWT095115) and the NIH (EB002520, DE016525) for financialsupport.
Received: November 17, 2013; Published online: DOI: 10.1002/mabi.201300513

Keywords: alkaline phosphatase; mineralization; osteogenesis;scaffolds; silk

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Enzymatic Mineralization of Silk Scaffolds