In Situ Evaluation of Calcium Phosphate Nucleation Kinetics and

Subscriber access provided by La Trobe University Library

Article

In situ evaluation of calcium phosphate nucleation kinetics and pathways during intra and extrafibrillar mineralization of collagen matrices Doyoon Kim, Byeongdu Lee, Stavros Thomopoulos, and Young-Shin Jun Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00864 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on August 1, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

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

Crystal Growth & Design

Cover Page In situ evaluation of calcium phosphate nucleation kinetics and pathways during intra and extrafibrillar mineralization of collagen matrices Doyoon Kim,† Byeongdu Lee,‡ Stavros Thomopoulos,§ and Young-Shin Jun*,† †

Department of Energy, Environmental & Chemical Engineering, Washington University, St. Louis,

Missouri 63130, United States ‡

X-ray Science Division, Argonne National Laboratory, Argonne, Illinois 60439, United States

§

Department of Orthopedic Surgery, Columbia University, New York, New York 10032-3072, United States

*Corresponding Author: Phone) 314-935-4539; Fax) 314-935-7211; E-mail) [email protected]; http://encl.engineering.wustl.edu/

ABSTRACT We revealed that nucleation sites within collagen fibrils determined pathways for calcium phosphate nucleation and its transformation, from amorphous species to crystalline plates, during the biomineralization process. Using in situ small angle X-ray scattering (SAXS), we examined the nucleation and growth of CaP within collagen matrices and elucidated how a nucleation inhibitor, polyaspartic acid (pAsp), governs mineralization kinetics and pathways at multiple length scales. Mineralization without pAsp led initially to spherical aggregates of CaP in the entire extrafibrillar spaces. With time, the spherical aggregates transformed into plates at the outermost surface of the collagen matrix, preventing intrafibrillar mineralization inside. However, mineralization with pAsp led directly to the formation of intrafibrillar CaP plates with a spatial distribution gradient through the depth of the matrix. The results illuminate mineral nucleation kinetics and real-time nanoparticle distributions within organic matrices in solutions containing body fluid components. Because the macroscale mechanical properties of collagen matrices depend on their mineral content, phase, and arrangement at the nanoscale, this study contributes to better design and fabrication of biomaterials for regenerative medicine.

ACS Paragon Plus Environment


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

1

In situ evaluation of calcium phosphate nucleation kinetics and

2

pathways during intra and extrafibrillar mineralization of collagen

3

matrices

4 Doyoon Kim,† Byeongdu Lee,‡ Stavros Thomopoulos,§ and Young-Shin Jun*,†

5 6 7

†

8

St. Louis, Missouri 63130, United States

9

‡

Department of Energy, Environmental & Chemical Engineering, Washington University,

X-ray Science Division, Argonne National Laboratory, Argonne, Illinois 60439, United

10

States

11

§

12

3072, United States

13

*Corresponding author

Department of Orthopedic Surgery, Columbia University, New York, New York 10032-

14 15

ABSTRACT

16

We revealed that nucleation sites within collagen fibrils determined pathways for calcium

17

phosphate nucleation and its transformation, from amorphous species to crystalline

18

plates, during the biomineralization process. Using in situ small angle X-ray scattering

19

(SAXS), we examined the nucleation and growth of CaP within collagen matrices and

20

elucidated how a nucleation inhibitor, polyaspartic acid (pAsp), governs mineralization

21

kinetics and pathways at multiple length scales. Mineralization without pAsp led initially

22

to spherical aggregates of CaP in the entire extrafibrillar spaces. With time, the spherical

23

aggregates transformed into plates at the outermost surface of the collagen matrix,

1 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

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


24

preventing intrafibrillar mineralization inside. However, mineralization with pAsp led

25

directly to the formation of intrafibrillar CaP plates with a spatial distribution gradient

26

through the depth of the matrix. The results illuminate mineral nucleation kinetics and

27

real-time nanoparticle distributions within organic matrices in solutions containing body

28

fluid components. Because the macroscale mechanical properties of collagen matrices

29

depend on their mineral content, phase, and arrangement at the nanoscale, this study

30

contributes to better design and fabrication of biomaterials for regenerative medicine.

31 32

INTRODUCTION

33

Mammalian mineralized tissues are natural composites of fibrillar collagen proteins1,2 and

34

calcium phosphate (CaP) nanocrystals.3-5 The combination of a compliant component

35

(collagen) with relatively stiff inclusions (CaP crystals) provides a wide range of tissue-

36

level mechanical properties.6-8 This range includes stiff (but brittle) tooth enamel, made

37

mostly of mineral,3,5 and partially mineralized tendon-to-bone attachments with high

38

toughness.7,9,10 Type I collagen molecules self-assemble into a fibril with a periodic 67

39

nm banded pattern.1,2 There are 40 nm gap zones between neighboring collagen

40

molecules, providing nucleation sites for intrafibrillar mineralization (IM).4,11,12

41

Stabilization of pre-nucleation clusters (PNCs) is essential to IM by inhibiting CaP

42

nucleation in extrafibrillar spaces (extrafibrillar mineralization, EM).11,13-15 In addition to

43

physiologically existing non-collagenous proteins (NCPs), such as fetuin,11,13

44

polyaspartic acid (pAsp) has been shown to promote IM during the in vitro mimicking of

45

collagen mineralization.11,12,16 Controlling the nucleation sites and kinetics using EM

46

inhibitors, such as pAsp, can be an effective approach to achieving targeted mechanical



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

47

properties of both individual collagen fibrils at the nanometer scale and tissue constructs

48

at the millimeter scale.

49

Previous nanoscale observations relied on snapshot images of debris16 or selected

50

cross sections from matrices17 or individual fibrils,11 typically focusing on late in vitro

51

mineralization events. These studies required extensive sample preparation procedures

52

prior to imaging or were limited to cryogenic sample environments,11,16,17 and so could

53

not fully explore the in situ kinetics of nucleation and growth of CaP, which involve a

54

dynamic sequence of morphological changes with phase transformations. The phase

55

transformations include amorphous CaP phases as important intermediate products.15,18-22

56

To understand the mineralization of tissue-level scaffolds, the spatial distribution of CaP

57

within millimeter-sized collagen matrices needs to be evaluated from the nanoscale, as

58

mineral crystals nucleate and grow within collagen fibrils,11 through the macroscale,

59

where mineral crystals are widely distributed in the extrafibrillar spaces between collagen

60

fibrils arranged within the matrix.17,23 Prior approaches have not provided continuous

61

real-time information on the spatial distribution of CaP within a collagen matrix over

62

these length scales. Without in situ kinetic evaluation of mineral nucleation and growth,

63

and of the spatial distribution of the mineral crystals at multiple length scales, a precise

64

design of CaP-stiffened collagen matrices cannot be achieved.

65

Studies utilizing synchrotron X-ray techniques have revealed important aspects of

66

bone and bioengineered materials.18,24-28 Recent studies have provided full three-

67

dimensional reconstruction of collagen fiber orientation in a human trabecula bone and

68

tooth, using small-angle X-ray scattering (SAXS) with tensor tomography.29,30 However,

69

in situ nucleation kinetics during collagen mineralization have not been evaluated despite 3 ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33

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


70

their importance in governing the chemical and mechanical properties of organic-

71

nanocrystal composites. Here, using SAXS, we provide in situ investigations31,32 of CaP

72

development (i.e., its morphology and distribution) within reconstituted collagen matrices

73

under the influence of pAsp, which controls the nucleation kinetics of CaP in

74

extrafibrillar space.11,12,33 This evaluation of the temporal development of CaP

75

demonstrates different pathways for intra and extrafibrillar mineralization. The

76

macroscale spatial distributions of CaP nanocrystals in different stages emphasize the

77

role of the extrafibrillar spaces of the collagen matrices in transport of nucleation and

78

growth precursors during tissue mineralization.

79 80

EXPERIMENTAL SECTION

81

The Experimental Section in the Supporting Information (SI) provides full details of the

82

preparation of samples and simulated body fluid, and descriptions of the flow-through

83

reactor, in situ X-ray scattering data collection and analysis, and ex situ analyses of

84

collagen mineralization.

85

Preparation of samples. Collagen matrices were reconstituted from Type I collagen34

86

(C857, calf skin lyophilized, Elastin Products Company, Inc.) in a specially designed

87

polytetrafluoroethylene (PTFE) frame (Figure 1a). The cover glass on both sides of the

88

frame held the solution and collagen, and also allowed X-ray penetration during the

89

SAXS analysis. The frame was 2 mm thick in the direction of the X-ray path. To

90

reconstitute the lyophilized collagen in a well-controlled shape with uniform thickness

91

for the SAXS evaluations, a collagen fibrillar density of 12 mg ml-1 was used. With this

92

collagen fibrillar density, we were able to observe both intra and extrafibrillar



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

93

mineralization behaviors clearly in the mineralization solution. In addition, thin collagen

94

films were prepared on glass slides to simulate the reactions at the outermost surface of

95

collagen matrices. CaP structures formed during collagen mineralization were compared

96

with synthetic hydroxyapatite (ACROS Organics™).

97

Simulated body fluid and the flow-through reactor. To simulate physiologically-

98

relevant conditions, while conducting our experiments within a reasonable time, a

99

simulated body fluid (SBF)35,36 with three times higher concentrations of calcium and

100

phosphate ions (3×SBF, pH 7.25) was used as an experimental solution (Table S1).

101

Either 0 or 10 mg l-1 of polyaspartic acid, pAsp (sodium salt, Mw: 5,000 Da, LANXES)

102

was added to the solution, depending on the experimental condition. To prevent any

103

precipitation prior to the experiment, the 3×SBF solutions containing either Ca or P

104

sources were prepared separately, and then combined just before the reaction, using a

105

syringe pump (Figure S1a). The reactor was maintained at 37±1 oC.

106

In situ X-ray scattering data collection and analysis. SAXS data were collected at the

107

Advanced Photon Source (APS, Sector 12 ID-B, Figure S1b) at Argonne National

108

Laboratory (Argonne, IL, USA).31,32 At intervals of 5, 9.5, and 15 h, frames were

109

immediately moved to the SAXS sample stage and scanned from z = 0–1.5 mm depth of

110

the matrix (Figure 1b). The distance from the sample to the SAXS detector was 3.6 m,

111

which provided a range of 0.0017–0.53 Å-1 for the scattering vector, q. For each scan, the

112

sample was exposed to a 14 keV X-ray beam for 1 s. The size of the beam was 150 µm

113

(perpendicular to the z-direction) × 40 µm (parallel to the z-direction). Two different

114

lateral positions were scanned at each time for duplicated samples; therefore, a total of

115

four scattering patterns were obtained for each condition and position. Control SAXS 5 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

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


116

intensities were acquired for unmineralized scenarios and used to define the background

117

intensity (Figure S2). The procedures for the data analysis are described in the SI

118

Experimental Section in detail.37-41

119

Similarly, in situ wide angle X-ray scattering (WAXS) analysis was conducted at

120

APS sector 11-ID-B to identify the CaP phases during collagen mineralization (q > 0.6

121

Å-1). Samples were exposed to a 58.66 keV X-ray beam (Size: 300 µm × 300 µm) for 5

122

min for the data collection.

123

Ex situ analyses of collagen mineralization. Thin sections (100 nm) of the collagen

124

matrices were prepared for TEM analysis. Matrices were fixed in 100 mM cacodylate

125

buffer containing 2% paraformaldehyde and 2.5% glutaraldehyde, followed by

126

dehydration in successive ethanol baths. Then samples were embedded in epoxy resin

127

and thin sections were prepared using an ultramicrotome (EM UC7, Leica). Osmium

128

tetroxide, uranyl acetate, and Reynold’s lead citrate were used for staining for TEM

129

imaging if needed. Two different instruments were used for TEM imaging. A JEOL 1200

130

EX II was operated at 100 kV, and a JEOL JEM-2100F was operated at 200 kV for

131

higher resolution images.

132

Collagen matrices and films were analyzed by Raman spectroscopy (inVia Raman

133

spectrometer, Renishaw plc) and scanning electron microscopy (SEM) equipped with

134

energy-dispersive X-ray spectroscopy (EDS) (FEI Nova NanoSEM 2300). Samples were

135

stored in an ethanol bath for more than 5 h to remove excess ions after mineralization.

136

The matrices were then frozen in liquid nitrogen and cut into ~1 mm thick sections

137

(Figure S6a).



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

138

Fluorescence correlation spectroscopy (FCS) was used to evaluate the

139

extrafibrillar pore structures of the matrices by measuring the diffusion time of

140

rhodamine 6G (R6G).42 We used an inverted design confocal laser scanning microscope

141

(Zeiss Axiovert 200M) equipped with a 40× water immersion lens and a 514 nm argon

142

laser. Fluorescence intensities were collected for 30 s × 10 times, then the diffusion time,

143

߬஽ , was evaluated by analyzing correlation curves.43

144 145

RESULTS AND DISCUSSION

146

SAXS analysis of CaP development within collagen matrices. An overview of the

147

influence of pAsp on CaP development can be seen from invariant values which are

148

proportional to the total volume of newly formed CaP particles with the same

149

morphology and structure at a given time.31,44 Invariant values, Q, are expressed as Q =

150

(1/2π2)∫q2I(q)dq, where I(q) is the scattering intensity at the scattering vector, q.37 As

151

shown in Figure 2a, for mineralized collagen matrices without pAsp (MC0p), Q increased

152

monotonically up to 15 h, without any variation in the spatial distribution at z = 0.3–1.5

153

mm. However, the outermost surface of the matrix (z = 0) showed Q values at 15 h more

154

than twice as high as those of other positions, with a statistically significant difference

155

(Figures 2b and S3a). In contrast, CaP formation was strongly hindered until 9.5 h in the

156

matrices mineralized with the addition of 10 mg l-1 pAsp (MC10p) as a nucleation

157

inhibitor.11,45 With pAsp, interestingly, a clear gradient in CaP distribution, with

158

statistically significant differences, was observed throughout the depth of the matrix at 15


Page 8 of 33

Page 9 of 33

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


159

h, demonstrating a direct relationship between the proximity of the SBF–matrix interfaces

160

and CaP formation (Figures 2c and S3b).

161

The analysis of individual SAXS patterns provided detailed information on the

162

evolution of CaP population and morphology.37,38 All SAXS profiles at 5–9.5 h,

163

regardless of the spatial position or inclusion of pAsp, had similar patterns with short

164

plateaus at small q, followed by a linear decrease in intensity (Figure 3). Those SAXS

165

patterns can be fit using the global unified scattering function, I(q) = G exp(-q2Rg2/3) +

166

Bq-P, to estimate the radius of gyration, Rg, from Guinier’s law, and the power-law

167

scattering exponent, P, from the slope at linear power-law regimes.37 In the absence of

168

pAsp (MC0p), the power-law scattering with an exponent of P ~ 3 is observed, which

169

indicates the existence of fractal structures. For mass fractal structures, such as loosely-

170

packed aggregates, P equals the fractal dimension Df.46 The power-law is terminated at

171

the lower q side by the Guinier type of scattering, indicating that the fractal structure is

172

finite in size. Combining these observations, we modeled this structure as a spherical

173

(isometric shape, Df = 3) aggregate with a fractal inner structure. Non-isometric objects,

174

such as cylinder or disk, would show a power-law slope less steep than -3, and a fractal

175

nature (either mass or surface) or surface roughness would reduce the slope further. From

176

the model, the size and population of the aggregates can be determined. The scattering

177

intensity from this population increased with time at all z positions, which resulted in a

178

clear increase in the volume fraction of spherical aggregates within the matrix (Vsp,

179

Figure 4a).

180

The intensities at high q for 15 h of mineralization showed typical SAXS patterns

181

for plate-like objects (the slope at q around 0.01–0.1 Å-1 is about -2, Porod scattering 8 ACS Paragon Plus Environment


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

182

appears at q above 0.2 Å-1),15,38 both for samples mineralized with pAsp (at all z

183

positions) and without pAsp (only at z = 0). These data can be fit using a specific form

184

factor and size distribution function (SI Experimental Section eq. S4–7). In the MC0P

185

system, a population of plates was observed only at the SBF–MC0p interface (z = 0) at

186

15 h (Figure 3a), showing a sudden, but significant, volume fraction of plates at that

187

location (Vpl, Figure 4b). Contrarily, populations of plates developed at all z-positions of

188

the MC10p samples at 15 h, with decreasing Vpl along the z-direction (Figure 3b and 4b).

189

The morphology of these plates (a thickness of 1.5 nm and mean diameter of 40 nm) was

190

comparable to widely accepted dimensions for apatite crystals in mature bone (~2.1 × 30

191

× 40 nm3).7,14 A similar thickness, though slightly thinner than that of crystals in mature

192

bone, was reported from a SAXS pattern of octacalcium phosphate-like plates (~1.4

193

nm),15 and most bone crystals observed were less than 2 nm thick.5,47 In our observations,

194

CaP plates developed from both MC0p and MC10p had almost identical thicknesses, and

195

the thickness of plates was uniform along the z-directions of MC10p. Another population

196

of spheres, following Porod’s law (P ~ 4), was also observed in MC10p samples,

197

demonstrating that this population was not formed by aggregation of smaller precursors

198

but consisted of individual compact spherical particles (Figure 3b and S3d,e). In addition,

199

Vsp was significantly lower in MC10p samples than in MC0p samples, without showing a

200

clear increasing trend over time until the development of plate-like crystals (Figure 4a).

201

The three different populations of particles with dissimilar morphologies and size

202

ranges observed by in situ SAXS analysis in this study show that CaP developments in

203

intra and extrafibrillar spaces undergo different pathways, although both pathways

204

include 1.5 mm thick plates at 15 h. The following discussion describes how those


Page 10 of 33

Page 11 of 33

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


205

pathways are determined by the nucleation inhibitor, pAsp, and how they consequently

206

influence the mineralization patterns of tissue-level constructs.

207 208

Distinctively different pathways for CaP mineralization in the intra and

209

extrafibrillar spaces of collagen matrices. A TEM image of thin sections of MC0p

210

indicated that CaP developed primarily in extrafibrillar spaces. At 15 h of mineralization,

211

aggregated thin plates (dashed circle and inset image in Figure 5a) were imaged near z =

212

0, around unmineralized collagen fibrils (black arrow in Figure 5a) showing brighter

213

contrast than the embedding epoxy resin. On the other hand, CaP development with 10

214

mg l-1 pAsp (MC10p) represents an IM-dominant system, showing darker contrasts from

215

collagen fibrils than the surrounding epoxy resin (white arrow in Figure 5b). The

216

different mineralization patterns resulting from pAsp were also evaluated from the thin

217

collagen films on glass slides after 15 h of mineralization (Figure 6). This process

218

simulated reactions at the outermost surface of the matrices (z = 0), which were not

219

imaged from thin sections cut along the z-direction. In the absence of pAsp, the collagen

220

surface was mostly covered with micrometer scale spheres, consisting of a number of

221

nanoscale plates (Figure 6a). The Raman spectra from these spheres is similar to that of

222

synthetic hydroxyapatite, with a clear phosphate peak at ∆960 cm-1 (the P–O band for

223

apatite, Figure 6d). This peak indicates CaP formed during the mineralization without

224

pAsp (Figure 6a), but the spectrum does not include peaks from collagen fibrils. On the

225

other hand, individual fibrils were clearly mineralized by the addition of 10 mg l-1 pAsp

226

(Figure 6b, the atomic percentages of Ca and P by SEM-EDS were 21.4 and 16.6,

227

respectively) losing their periodic patterns shown in the unmineralized fibrils (Figure 5c 10 ACS Paragon Plus Environment


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

228

and 6c). In Raman spectra, peaks indicating both apatite (at ∆960 cm-1 for the P–O band)

229

and collagen (at ∆2,941cm-1 for the C-H band) clearly appeared (Figure 6d). Similar

230

Raman spectra have been frequently reported from partially mineralized tendons or bone

231

samples.9,10 Ca/P molar ratios of EM and IM in this study were 1.19 and 1.29. Those

232

numbers are slightly lower than the theoretical value of octacalcium phosphate (1.33),

233

which are expected to appear during the early stage CaP development process.15

234

Based on ex situ observations (Figures 5 and 6), we concluded that the addition of

235

10 mg l-1 pAsp effectively controlled the IM and EM patterns. This control allowed us to

236

evaluate the nucleation kinetics and pathways for both mineralization processes

237

separately, using in situ SAXS observations. To better evaluate the different nucleation

238

kinetics and pathways during both mineralization processes and to thoroughly investigate

239

this complex system, we simplified the system to use one type and concentration of pAsp

240

(10 mg l-1 of 5,000 Da pAsp).

241

From the SAXS analyses during EM (MC0p), we found a CaP population of

242

spherical aggregates (Figure 3a, P ~ 3) as intermediate products. This finding is

243

consistent with a CaP nucleation pathway without collagen fibrils in the absence of

244

nucleation inhibitors, as observed in recent cryogenic studies of CaP formation.15,20 These

245

studies showed that amorphous CaP spheres formed via aggregation and densification of

246

PNCs before transformation into plate-like morphologies with increased crystallinity. The

247

diameters of the densified aggregates in prior reports were 30–80 nm,15,20 consistent with

248

the SAXS results of the current study (Rg = 20.5–32.3 nm, equivalent to a diameter =

249

52.9–83.4 nm, Figure S3f). A decreasing pattern in Rg over time can be evidence of the

250

CaP pathway of the densification of ionic clusters as nucleation precursors. 11 ACS Paragon Plus Environment

Page 12 of 33

Page 13 of 33

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


251

During the CaP development with pAsp (MC10p), plate-like crystals (akin to the

252

shape of bone apatite crystals) formed without an intermediate phase of spherical

253

aggregates (between 9.5 and 15 h, Figure 3b), and this showed that the pathway of CaP

254

development for IM is clearly distinguished from that for EM. This new finding also

255

indicates that CaP transformation to plates for IM is much faster (once nucleation occurs)

256

than for EM. In the absence of pAsp, it took 15 h for the transformation of spherical

257

aggregates to plates in the extrafibrillar spaces, and this transformation was observed

258

only at the top surface of the matrix (Figure 3a). Therefore, the inhibition of CaP

259

nucleation at the extrafibrillar spaces by pAsp allows the nucleation in the intrafibrillar

260

spaces, and during this IM process, CaP takes a new pathway, which is faster in its

261

transformation but slower in nucleation than EM process.

262

Similarly, in situ WAXS data show that crystalline structure of CaP developed

263

faster in MC10p than in MC0p (Figure S4). The WAXS pattern clearly shows peaks

264

(corresponding to the (002), (211), and (112) faces of hydroxyapatite) developed in

265

MC10p after 20 h. When CaP developed in the extrafibrillar structure (MC0p), only the

266

(002) peak appeared, indicating that more amorphous or poorly crystalline states

267

persisted for a longer period. This finding supports our SAXS observation that the

268

morphological transformation from spherical aggregates to plates in MC0p was

269

maintained for a relatively longer period (compared to the direct development of plates

270

after the incubation time in MC10p) and was limited to only the outermost surface.

271

On the other hand, the spherical population observed for MC10p (Compact

272

spherical particles, P ~ 4) does not seem to be directly related to plates, due to its large

273

size (Rg = 47–71 nm, Figure S3f) and much lower Vsp than Vpl (Figure 4a,b). Instead, CaP 12 ACS Paragon Plus Environment


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

274

spheres might develop from aqueous Ca-pAsp complexation in the extrafibrillar space.13

275

However, the SAXS patterns that are fitted to compact spheres had minimal influence on

276

the fitting of plate-like crystals (Figure S3d), validating that plate-like CaP developed

277

without any intermediate products.

278

In situ SAXS and WAXS observations indicate that nanoscale evaluations of

279

amorphous CaP phase are critical to thoroughly understand the kinetics and pathways of

280

CaP nucleation and its phase transformation. This study highlights the importance of in

281

situ X-ray scattering applications for future studies on the early stage collagen

282

mineralization.

283 284

Macroscale spatial distribution of CaP nanocrystals. Despite the importance of

285

collagen fibrillar structure on tissue mineralization, as suggested in recent studies,17,48,49

286

the role of extrafibrillar space, which can be an important entryway for molecules

287

required for the CaP mineralization, has been neglected in most studies. Therefore,

288

researchers have often not connected the implications from in vitro findings to those

289

found in vivo. The spatial distribution of CaP as a function of the distance from the SBF–

290

matrix interface explored in this study is especially important for understanding how

291

molecules, such as PNCs or Ca2+ ions, are delivered from the SBF to the inside of the

292

matrix to form CaP plates. The occupation of extrafibrillar pores with CaP (Figures 5a

293

and 6a) could influence the transport of those molecules within the matrix by limiting

294

their diffusion.42


Page 14 of 33

Page 15 of 33

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


295

To examine this possibility, the diffusion time, ߬஽ , of rhodamine 6g (R6G) was

296

evaluated using fluorescence correlation spectroscopy (Figure 7a). ߬஽ at z = 0 was about

297

three times higher than at other positions in MC0p at 15 h, indicating that a diffusion of

298

molecules decreased, especially at the SBF–matrix interface, and further prevented

299

nucleation precursors from penetrating into the deeper positions of the matrix. The

300

increased diffusion time corresponds to the SAXS observation that the increase in

301

scattering intensity from MC0p became mild at z = 0.3–1.5 mm (Figure 2a). In

302

consequence, a thin layer of aggregated plates formed on the outer surface of MC0p in a

303

later stage, as clearly imaged using TEM (Figures 7b and S5a-c). These aggregated plates

304

filled the extrafibrillar space less than 0.2 mm deep from the top surface, but they were

305

scarcely observed at deeper z-positions. This layer could be a diffusion barrier for the

306

further nucleation in deeper positions of MC0p. Ex situ analyses of matrices’ sections

307

using SEM and Raman spectroscopy demonstrated that IM was not achieved in MC0p

308

even after the additional 23 h mineralization period, showing clearly distinguishable

309

features between the extrafibrillar CaP layer and collagen matrix (Figure S6 and Table

310

S2).

311

On the other hand, the increase in ߬஽ after mineralization in MC10p was

312

relatively low compared to MC0p (Figure 7a), demonstrating that neither the occupation

313

of extrafibrillar spaces by CaP crystals nor the influence of the intrafibrillar CaP plate on

314

the extrafibrillar space was significant, allowing scattering intensities to increase with a

315

relatively steep slopes at low z positions of MC10p (Figure 2a). Therefore, CaP plates can

316

form more deeply in the matrices even at z = 1.5 mm (Figure 4b). The extrafibrillar CaP

317

crystals in MC10p were visible only at the later stage, showing elongated morphologies



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

318

in micrometer scale (Figures 7c and S5d-f). Unlike in MC0p, extrafibrillar CaP crystals

319

were not severely aggregated (corresponding to different P values for spherical

320

populations in MC0p and MC10p, Figure S3e) and did not significantly occupy the

321

extrafibrillar spaces except for a few areas at z = 0 (Figure 7c).

322 323

Kinetic control of CaP nucleation for tissue-level mineralization. The proposed

324

mechanisms of CaP development in collagen matrices are described in Figure 8. In the

325

absence of a nucleation inhibitor, spherical aggregates form in extrafibrillar spaces, in a

326

fashion similar to CaP developing in solution.15,20 Initially, CaP precursors, such as PNCs

327

or other ionic Ca and P species, move freely within the matrix through the relatively large

328

extrafibrillar spaces, nucleating in the extrafibrillar spaces with little z-position

329

dependency. This population of spherical aggregates grows continuously, filling the

330

extrafibrillar spaces while reducing the diffusion of precursors penetrating deeper into the

331

matrix. Over time, spherical aggregates require continuously bond with Ca2+ from the

332

solution and transform into crystalline plates,15 but this dominantly occurs only at the

333

solution–matrix interface (Figure 4b). These aggregated plates eventually become dense

334

enough to create a diffusion barrier at the later stage (Figure 7b), inhibiting the transport

335

of precursors to the inside of the matrix and preventing further mineralization throughout

336

its depth.

337

In the presence of a nucleation inhibitor (representing the physiologically relevant

338

case), the nucleation of CaP in extrafibrillar spaces is kinetically restricted by aqueous

339

complexation of Ca2+ ions with inhibitors. Thus, PNCs or other CaP precursors can be


Page 16 of 33

Page 17 of 33

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


340

continuously supplied to collagen gap channels before they are consumed for EM or

341

blocked by a diffusion barrier. The confined space of narrow gap channels (40 nm wide,

342

but less than 2 nm high2,5,6) does not allow for the formation of large aggregates; hence,

343

nuclei can directly grow as plates without the intermediate step of spherical aggregates,

344

as observed in MC0P. When ionic precursors do not form aggregated clusters due to the

345

inhibition by pAsp, the activation energy barrier for creating new surfaces for nucleation

346

of CaP in extrafibrillar spaces cannot be overcome easily.15 Therefore, about 9.5 h of

347

induction time was required before the initial development of CaP plates. Once

348

nucleation occurs within the gap channels, however, CaP nuclei strongly attract

349

precursors and begin to grow into plates. This pathway results in a faster morphological

350

transformation (Figure 3) and crystallization (Figure S4) than the pathway with

351

intermediate spherical aggregates in the absence of pAsp. The consumption of precursors

352

by nuclei for IM cause the delivery of fewer precursors to deeper z-positions, therefore,

353

intrafibrillar CaP plates develop with a gradual spatial distribution.

354 355

CONCLUSIONS

356

In summary, we evaluated the kinetics of mineralization of collagen matrices at nano

357

through macroscales, using in situ SAXS analysis. The evolution of CaP’s morphology

358

and its volume size distribution were separately determined during nucleation and growth

359

within extra and intrafibrillar spaces. The findings of this study revealed that the

360

pathways and kinetics of CaP development during intrafibrillar mineralization are

361

distinctively different from those during extrafibrillar CaP mineralization. In the absence



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

362

of nucleation inhibitors, improper control of CaP nucleation kinetics brought significant

363

CaP populations of spherical aggregates in the extrafibrillar spaces. This highlights the

364

importance of including nucleation inhibitors for recreating physiologically relevant

365

mineralization of collagen-based matrices.11,12

366

In situ SAXS measurement at the different z-positions utilized in this study has a

367

number of practical benefits. For example, we were able to detect unexpected layers that

368

clearly formed at the outermost surface (z = 0), which disrupts the preparation of

369

biomaterials with a uniform mineral distribution in macroscale. The quantitative

370

information showing gradual development of plate-like crystals along the z-positons can

371

be adapted to evaluate the kinetic parameters, such as the diffusion coefficients of

372

precursor molecules for the nucleation of biominerals in biological templates.

373

Furthermore, because the distribution of CaP minerals relative to collagen, at both nano

374

and macroscales, dictates the mechanics of the tissue,7,8 the current results provide

375

guidance for proper mineralization of scaffolds for creating in vitro model systems and

376

for developing constructs for regenerative medicine.

377 378

ASSOCIATED CONTENT

379

Supporting Information

380

Full details of Experimental Section. Background SAXS intensities and additional SAXS

381

patterns and their fittings from mineralized collagen matrices. WAXS patterns from

382

mineralized collagen matrices. Characterizations of sections of collagen matrices (TEM


Page 18 of 33

Page 19 of 33

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


383

and SEM images and Raman spectra). This material is available free of charge via the

384

Internet at http://pubs.acs.org.

385 386

AUTHOR INFORMATION

387

Corresponding Author

388

*[email protected]

389 390

ACKNOWLEDGEMENTS

391

The authors acknowledge the Biomineralization discussion group at Washington

392

University in St. Louis, consisting of J. D. Pasteris, G. Genin, T. Daulton, A. Deymier, A.

393

G. Schwartz, J. Lipner, and J. Boyle for helpful discussions. We thank J. Ballard for

394

carefully reviewing the manuscript. The project was mainly supported by Y.S.J.’s faculty

395

startup fund at Washington University and partially supported by the National Science

396

Foundation (DMR-1608545 and DMR-1608554) and the National Institutes of Health

397

(U01 EB016422). The Nano Research Facility, Institute of Materials Science &

398

Engineering, Molecular Microbiology Imaging Facility, Confocal Microscopy Facility,

399

and Soft Nanomaterials Laboratory at Washington University in St. Louis provided their

400

facilities for the experiments. Use of the Advanced Photon Source (sector 12 ID-B and

401

11ID-B) at Argonne National Laboratory was supported by the U.S. Department of

402

Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-

403

AC02-06CH11357.



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

404 405

REFERENCES

406

(1) Hodge, A. J.; Petruska, J. A. In Aspects of protein structure (ed. G. N.

407

Ramachandran); Academic Press: New York, NY, 1963; pp 289–300.

408

(2) Orgel, J. P. R. O.; Irving, T. C.; Miller, A.; Wess, T. J. Proc. Natl. Acad. Sci. U.S.A.

409

2006, 103, 9001-9005.

410

(3) Glimcher, M. J. Rev. Mineral. Geochem. 2006, 64, 223-282.

411

(4) Landis, W.; Jacquet, R. Calcif. Tissue Int. 2013, 93, 329-337.

412

(5) Pasteris, J. D.; Wopenka, B.; Valsami-Jones, E. Elements 2008, 4, 97-104.

413

(6) Alexander, B.; Daulton, T. L.; Genin, G. M.; Lipner, J.; Pasteris, J. D.; Wopenka, B.;

414

Thomopoulos, S. J. R. Soc. Interface 2012, 9, 1774-1786.

415

(7) Liu, Y.; Thomopoulos, S.; Chen, C.; Birman, V.; Buehler, M. J.; Genin, G. M. J. R.

416

Soc. Interface 2014, 11.

417

(8) Nair, A. K.; Gautieri, A.; Chang, S.-W.; Buehler, M. J. Nat. Commun. 2013, 4, 1724.

418

(9) Schwartz, A. G.; Pasteris, J. D.; Genin, G. M.; Daulton, T. L.; Thomopoulos, S. PLoS

419

One 2012, 7, e48630.

420

(10) Wopenka, B.; Kent, A.; Pasteris, J. D.; Yoon, Y.; Thomopoulos, S. Appl. Spectrosc.

421

2008, 62, 1285-1294.

422

(11) Nudelman, F.; Pieterse, K.; George, A.; Bomans, P. H. H.; Friedrich, H.; Brylka, L.

423

J.; Hilbers, P. A. J.; de With, G.; Sommerdijk, N. A. J. M. Nat. Mater. 2010, 9, 1004-

424

1009.

425

(12) Olszta, M. J.; Cheng, X.; Jee, S. S.; Kumar, R.; Kim, Y.-Y.; Kaufman, M. J.;

426

Douglas, E. P.; Gower, L. B. Mater. Sci. Eng., R 2007, 58, 77-116.


Page 20 of 33

Page 21 of 33

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


427

(13) Heiss, A.; DuChesne, A.; Denecke, B.; Grötzinger, J.; Yamamoto, K.; Renné, T.;

428

Jahnen-Dechent, W. J. Biol. Chem. 2003, 278, 13333-13341.

429

(14) Landis, W. J.; Song, M. J.; Leith, A.; McEwen, L.; McEwen, B. F. J. Struct. Biol.

430

1993, 110, 39-54.

431

(15) Habraken, W. J. E. M.; Tao, J.; Brylka, L. J.; Friedrich, H.; Bertinetti, L.; Schenk, A.

432

S.; Verch, A.; Dmitrovic, V.; Bomans, P. H. H.; Frederik, P. M.; Laven, J.; van der

433

Schoot, P.; Aichmayer, B.; de With, G.; DeYoreo, J. J.; Sommerdijk, N. A. J. M. Nat.

434

Commun. 2013, 4.

435

(16) Jee, S. S.; Culver, L.; Li, Y.; Douglas, E. P.; Gower, L. B. J. Cryst. Growth 2010,

436

312, 1249-1256.

437

(17) Wang, Y.; Azaïs, T.; Robin, M.; Vallée, A.; Catania, C.; Legriel, P.; Pehau-

438

Arnaudet, G.; Babonneau, F.; Giraud-Guille, M.-M.; Nassif, N. Nat. Mater. 2012, 11,

439

724-733.

440

(18) Betts, F.; Posner, A. S. Mater. Res. Bull. 1974, 9, 353-360.

441

(19) Combes, C.; Rey, C. Acta Biomater. 2010, 6, 3362-3378.

442

(20) Dey, A.; Bomans, P. H.; Müller, F. A.; Will, J.; Frederik, P. M.; Sommerdijk, N. A.

443

Nat. Mater. 2010, 9, 1010-1014.

444

(21) Mahamid, J.; Aichmayer, B.; Shimoni, E.; Ziblat, R.; Li, C.; Siegel, S.; Paris, O.;

445

Fratzl, P.; Weiner, S.; Addadi, L. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 6316-6321.

446

(22) Posner, A. S.; Betts, F. Acc. Chem. Res. 1975, 8, 273-281.

447

(23) Landis, W. J.; Hodgens, K. J.; Song, M. J.; Arena, J.; Kiyonaga, S.; Marko, M.;

448

Owen, C.; McEwen, B. F. J. Struct. Biol. 1996, 117, 24-35.

449

(24) Campi, G.; Ricci, A.; Guagliardi, A.; Giannini, C.; Lagomarsino, S.; Cancedda, R.;

450

Mastrogiacomo, M.; Cedola, A. Acta Biomater. 2012, 8, 3411-3418.



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

451

(25) Campi, G.; Fratini, M.; Bukreeva, I.; Ciasca, G.; Burghammer, M.; Brun, F.;

452

Tromba, G.; Mastrogiacomo, M.; Cedola, A. Acta Biomater. 2015, 23, 309-316.

453

(26) Fratzl, P.; Fratzl-Zelman, N.; Klaushofer, K.; Vogl, G.; Koller, K. Calcif. Tissue Int.

454

1991, 48, 407-413.

455

(27) Fratzl, P.; Groschner, M.; Vogl, G.; Plenk, H.; Eschberger, J.; Fratzl-Zelman, N.;

456

Koller, K.; Klaushofer, K. J. Bone Miner. Res. 1992, 7, 329-334.

457

(28) Fratzl, P.; Schreiber, S.; Klaushofer, K. Connect. Tissue Res. 1996, 34, 247-254.

458

(29) Liebi, M.; Georgiadis, M.; Menzel, A.; Schneider, P.; Kohlbrecher, J.; Bunk, O.;

459

Guizar-Sicairos, M. Nature 2015, 527, 349-352.

460

(30) Schaff, F.; Bech, M.; Zaslansky, P.; Jud, C.; Liebi, M.; Guizar-Sicairos, M.; Pfeiffer,

461

F. Nature 2015, 527, 353-356.

462

(31) Jun, Y.-S.; Lee, B.; Waychunas, G. A. Environ. Sci. Technol. 2010, 44, 8182-8189.

463

(32) Li, Q.; Fernandez-Martinez, A.; Lee, B.; Waychunas, G. A.; Jun, Y.-S. Environ. Sci.

464

Technol. 2014, 48, 5745-5753.

465

(33) Olszta, M. J.; Douglas, E. P.; Gower, L. B. Calcif. Tissue Int. 2003, 72, 583-591.

466

(34) Boyle, J. J.; Kume, M.; Wyczalkowski, M. A.; Taber, L. A.; Pless, R. B.; Xia, Y.;

467

Genin, G. M.; Thomopoulos, S. J. R. Soc. Interface 2014, 11.

468

(35) Gamble, J. L. Chemical Anatomy Physiology and Pathology of Extracellular Fluid:

469

6th ed.; Harvard University Press: Cambridge, MA, 1967.

470

(36) Ohtsuki, C.; Kokubo, T.; Yamamuro, T. J. Non-Cryst. Solids 1992, 143, 84-92.

471

(37) Beaucage, G.; Kammler, H. K.; Pratsinis, S. E. J. Appl. Crystallogr. 2004, 37, 523-

472

535.

473

(38) Fratzl, P. J. Appl. Crystallogr. 2003, 36, 397-404.

474

(39) Ilavsky, J.; Jemian, P. R. J. Appl. Crystallogr. 2009, 42, 347-353.


Page 22 of 33

Page 23 of 33

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


475

(40) Zhang, F.; Ilavsky, J.; Long, G.; Quintana, J. G.; Allen, A.; Jemian, P. Metall. Mater.

476

Trans. A 2010, 41, 1151-1158.

477

(41) Beaucage, G. J. Appl. Crystallogr. 1995, 28, 717-728.

478

(42) Kihara, T.; Ito, J.; Miyake, J. PLoS One 2013, 8, e82382.

479

(43) Rigler, R.; Mets, Ü.; Widengren, J.; Kask, P. Eur. Biophys. J. 1993, 22, 169-175.

480

(44) Liu, J.; Pancera, S.; Boyko, V.; Shukla, A.; Narayanan, T.; Huber, K. Langmuir

481

2010, 26, 17405-17412.

482

(45) Tas, A. C. Acta Biomater. 2014, 10, 1771-1792.

483

(46) Li, T.; Senesi, A. J.; Lee, B. Chem. Rev. 2016, DOI:10.1021/acs.chemrev.5b00690.

484

(47) Eppell, S. J.; Tong, W.; Lawrence Katz, J.; Kuhn, L.; Glimcher, M. J. J. Orthop.

485

Res. 2001, 19, 1027-1034.

486

(48) Cantaert, B.; Beniash, E.; Meldrum, F. C. Chem. -Eur. J. 2013, 19, 14918-14924.

487 488

(49) Cantaert, B.; Beniash, E.; Meldrum, F. C. J. Mater. Chem. B 2013, 1, 6586-6595.



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

489

List of Figures

490

491 492

Figure 1. (a) Design of custom made sample frame containing collagen matrix between

493

two cover glasses, with an indication of the z-direction. (b) Experimental setup for SAXS

494

measurements. ki and kf are the incident and scattered wave vectors, respectively, and 2θf

495

is the exit angle of the X-rays. SAXS measurements were taken at different positions of

496

the matrix along the z-direction from 0 (the interface between the SBF and collagen

497

matrix) to 1.5 mm, with 0.3 mm steps.

498


Page 24 of 33

Page 25 of 33

Q

(a)

0z =mm 0 mm 0.3 mmmm z = 0.3 0.6 mmmm z = 0.6 0.9 mmmm z = 0.9 1.2 mmmm z = 1.2 1.5 mmmm z = 1.5

10-4

MC0p MC10p

10-5

10-6

0

Q

(b)

5

1.0x10-4

Col z =2 0 mm Col 3 z =4 0.3 mm Col Col z =5 0.6 mm Col 6 z =7 0.9 mm Col

0.6x10-4

z = 1.2 mm z = 1.5 mm

0.2x10-4

(c) 1.0x10-4

Q

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


0.6x10-4

Time (h)

10

MC0p

15 ** *

######

######

######

5

9.5 Time (h)

15

z =20 mm Col Col z =30.3 mm Col 4 z =50.6 mm Col Col z =60.9 mm Col 7

MC10p

z = 1.2 mm z = 1.5 mm

**

0.2x10-4 5

** * ** * ** #####

9.5 Time (h)

499

15

500

Figure 2. (a) Spatio-temporal development of CaP within collagen matrix, observed

501

from the invariant, Q, derived from the integration of SAXS intensities. The background

502

intensity from unmineralized collagen matrices was subtracted from the SAXS

503

intensities. (b-c) Statistical analysis of Q values from MC0p and MC10p, using a three-

504

factor analysis of variance and post-hoc t-tests. The asterisk symbols * and ** indicate

505

that an averaged Q has a significant difference (p < 0.05) compared to the next and

506

second next z-positions, respectively. A sharp symbol # indicates a significant difference

507

compared to a measurement at previous time interval.



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

508 509

Figure 3. SAXS patterns from collagen matrices and their fittings. SAXS patterns from

510

MC0p (a) and MC10p (b) over time at z = 0–0.6 mm, and their fittings. The SAXS

511

patterns fit one or two CaP populations with morphologies of spherical aggregates,

512

compact spheres, or plates. For example, the SAXS pattern at z = 0.3 mm of MC0p at 15

513

h fits spherical aggregates (Rg = 22.2 nm and P = 3.05), and the corresponding pattern of

514

MC10p fits a combination of compact spheres (Rg = 70.1 nm and P = 3.99, at small q)

515

and plates (diameter = 40 nm with a standard deviation of 0.15 in log-normal

516

distributions and thickness = 1.5 nm, at large q).

517


Page 26 of 33

Page 27 of 33

(a) MC0p MC10p

(b)

z = 0 mm z = 0.3 mm z = 0.6 mm

3 2 1

1.5

Vpl (10-3 cm3/cm3)

Vsp (10-3 cm3/cm3)

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


0 66

518

12 99 12 Time (hr)

1.0 0.5 0

15 15

MC0p MC10p

0 0.0

0.3 0.3 0.6 0.6 0.9 0.9 1.2 1.2 1.5 1.5 z (mm)

519

Figure 4. (a) Volume fractions of CaP populations with spherical morphologies, Vsp, at

520

5–15 h and (b) volume fractions of plate-like shapes, Vpl, at 15 h, determined from

521

fittings of SAXS patterns. No plate-like shape particles were identified from SAXS

522

patterns of MC0p below 0.3 mm. The volume fractions of each particle population were

523

estimated by calibrating I(q) using a glassy carbon reference sample.

524



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

525 526

Figure 5. TEM images of thin sections of collagen matrices. (a) Near the outer surface (z

527

= 0) of a MC0p sample at 15 h. The black arrow indicates an unmineralized collagen

528

fibril. The dashed circle surrounds an aggregate of extrafibrillar CaP plates. The inset is a

529

high resolution TEM image of extrafibrillar CaP plates, corresponding to the features in

530

the circle. (b) Near the outer surface (z = 0) of the MC10p at 15 h. The higher

531

magnification inset is taken from a neighboring area of the sample. Collagen fibrils,

532

indicated with a white arrow, are mineralized, showing darker contrast than the

533

surrounding epoxy resin. No staining was applied to collagen fibrils for (a) and (b)

534

because precipitates from staining cannot easily be distinguished from CaP particles

535

formed within the matrices. (c) Stained unmineralized collagen fibrils demonstrated a

536

clear banding pattern of collagen fibrils. 27 ACS Paragon Plus Environment

Page 28 of 33

Page 29 of 33

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


537



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

538 539

Figure 6. (a-c) SEM images of collagen films on glass. (a) Mineralized for 15 h in

540

3×SBF without pAsp. (b) Mineralized for 15 h in 3×SBF with 10 mg l-1 pAsp. (c)

541

Unmineralized collagen. (d) Raman spectra of (a-c) and synthetic hydroxyapatite for

542

comparison.

543


Page 30 of 33

Page 31 of 33

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


544 545

Figure 7. Reduced diffusion of R6G during the early stage (within 15 h) of extrafibrillar

546

mineralization results in a nucleation barrier in a later stage of collagen mineralization

547

(after additional 23 h). (a) Diffusion times of R6G into MC0p and MC10p at different

548

depths after 15 h of mineralization. The average and standard deviation were obtained

549

from 10 measurements at each scanning position. Two different positions were scanned

550

at the same depth. Diffusion times in unmineralized collagen matrix and simulated body

551

fluid without Ca2+ are also indicated as grey regions for the comparison. (b-c) Spatial

552

distribution of CaP within MC0p (b) and MC10p (c) at the late stage of the

553

mineralization influenced by the reduced diffusion of molecules required for the CaP

554

mineralization from SBF solution into the deeper positions of the matrices. After an

555

initial 15 h of mineralization, for in situ SAXS analysis, TEM images of thin sections

556

were taken after an additional 23 h incubation in 3×SBF. Scale bars are 5 µm.

557



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

558

559 560

Figure 8. Mechanisms of (a) extrafibrillar mineralization within collagen matrices in the

561

absence (MC0p) and (b) intrafibrillar mineralization of collagen fibrils in the presence of

562

pAsp (MC10P).

563


Page 32 of 33

Page 33 of 33

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


564

For Table of Contents Use Only

565

Manuscript title: In situ evaluation of calcium phosphate nucleation kinetics and

566

pathways during intra and extrafibrillar mineralization of collagen matrices.

567

Author list: Doyoon Kim,† Byeongdu Lee,‡ Stavros Thomopoulos,§ and Young-Shin

568

Jun*,†

569

570 571 572

Synopsis: In situ SAXS observations uncovers distinctive nucleation pathways and

573

kinetics for calcium phosphate minerals at extra- and intrafibrillar spaces of collagen

574

matrices. When polyaspartic acid is presence in the system, it prevents formation of the

575

spherical aggregates of calcium phosphate nucleation precursors as an intermediate

576

products, and it leads directly to intrafibrillar mineralization with plate-like calcium

577

phosphate.


In Situ Evaluation of Calcium Phosphate Nucleation Kinetics and

Recommend Documents