Hierarchical Microspheres Constructed from Chitin Nanofibers

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Hierarchical Microspheres Constructed from Chitin Nanofibers Penetrated Hydroxyapatite Crystals for Bone Regeneration Bo Duan, Kangquan Shou, Xiaojuan Su, Yahui Niu, Guan Zheng, Yao Huang, Ai-xi Yu, Yu Zhang, Hong Xia, and Lina Zhang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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Biomacromolecules

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HierarchicalMicrospheres ConstructedfromChitin

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Nanofibers Penetrated Hydroxyapatite Crystals for Bone

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Regeneration

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Bo Duana, Kangquan Shoub, Xiaojuan Sua, Yahui Niub, Guan Zhengc, Yao Huanga,

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Aixi Yub, Yu Zhangc, Hong Xiac, Lina Zhanga

8 9

a

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,

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China b

11 12 13

c

Zhongnan Hospital, Wuhan University, Wuhan 430072, China

Department of Orthopedics, General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou 510010, China

14 15 16 17 18 19 20 21 22 23 24 25 * To whom correspondence should be addressed. Phone: +86-27-87219274. Fax:+86-27-68754067.E-mail:,[email protected],(L. Zhang) 1 ACS Paragon Plus Environment

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Abstract

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Chitin exists abundantly in the crab and shrimp shell as the template of the

28

minerals, which inspired us to mineralize it for fabricating the bone grafting materials.

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In present work, chitin nanofibrous microspheres were used as the matrix for in situ

30

synthesisof the hydroxyapatite (HA) crystals including micro-flake, submicron-needle

31

and submicron-osphere, which were penetrated by long chitin nanofibers, leading to

32

the hierarchical structure. The shape and size of the HA crystals could be controled by

33

changing the HA synthesisprocess. The tight interface adhesionbetween chitin and

34

HAthrough the non covanlent bonds occurred in the composite microspheres, and

35

HAswere homogeneously dispersed and bounded to the chitin nanofibers.In our

36

findings, the inherent biocompatibilities of the both chitin and HA contributed

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bone cell adhesion and osteoconduction. Moreovere, the chitin microsphere with

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submicron-needle and submicron-sphere HA crystals remarkably promoted in vitro

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cell adhesion and in vivo bone healing. It was demonstrated that the rabbit with 1.5

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cm radius defect were almost cured completely within three months at a growth factor

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and cell free state, as a result of the uniquesurface microstructure. The microsphere

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scaffold displayed the excellent biofunctions and an appropriate biodegradability.

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This work opened up a new avenue to construct the natural polymer based

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organic-inorganic hybrid microspheres for the bone regeneration.

45 46

Keywords: Microsphere scaffold, Chitin nanofibers, Penetratedhydroxyapatite

47

crystals, Microchannels, Biocompatibility 2 ACS Paragon Plus Environment

the

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Biomacromolecules

Introduction

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The repair of critical size bone defects which occur after trauma, infection, or

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tumor resections is a clinical challenge due to the apparent inability of bone to

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regenerate themselves.1-6 The bone tissue engineering scaffolds mimicking the native

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bone extracellular matrix (ECM) have recently emerged as a promising class of

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materials for bone repair.7-8 As natural bone is mainly composed of collagen

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nanofibers and hydroxyapatite (HA) nanocrystals, the ECM-mimicking nanofibrous

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composition of biocompatible polymer and HA have been extensively demonstrated

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to be a successful alternative for bone tissue regeneration.9 The broadly used strategy

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for fabricating the polymer-ceramic composition is to process the nanocrystal

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dispersed polymer-containing solution with electrospinning,10 phase separation11-12

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and self-assembly13 methods into the required ECM-mimicking collagen nanofibrous

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structure. However, the polymer matrix would obstruct the direct contact between

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cells and HA, which might cripple the desired biofunctions such as high

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osteoconduction.14-15 In sharp contrast with the “embedded” method, the in-situ HA

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mineralization approach on the polymer substrate appears as an available pathway to

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directly introduce the osteoconduction biofunction to the scaffold materials, and

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expand further the scope of the resulting materials for targeting applications in bone

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scaffolding. However, the in-situ HA mineralization within an extracellular is known

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to be a very complex process, and the crystal might not nucleate on the synthetic

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polymer materials or inversely grow into an excessive large size and cover the 3 ACS Paragon Plus Environment

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nanofibrous surface completely, as a result hindering the biofunctions of the

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nanofibrous matrix (promote cell adhesion). In the natural bone hierarchical

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nanostructure, the noncollagenous proteins (NCPs) plays important role on mediating

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the homogeneous nucleation of hydroxyapatite crystals within the collagen matrix due

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to their high affinity to calcium cations.16 To mimic the natural biomineralization

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process, the organic molecules including polyacrylic acid,8 adenosine 5-triphosphate

75

(ATP),17etc have been used as the alternative of NCPs for adjusting the compatibility

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between the dissimilar organic and inorganic phases in the hybrids. Notably, in the

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natural shells of marine animals, both the nanofibrous chitin and protein are thought

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to play key roles in providing organic interfaces as templates for the hierarchical

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mineralized architecture formation,

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living organisms possesses excellent biocompatibility, biodegradability and

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nontoxicity.21-27It is worth noting that chitin has been extensively investigated as

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anadsorbent for metal extraction due to the chelation properties of the large amount

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ofacetyl amino groups to the metal ions.28-30 This indicates that the chitin matrix is an

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excellent support candidate, and has appropriate interface for stabilizingHA

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crystals.Meanwhile, the amphilic chitin could effectively avoid the excessive growth

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of the hydrophilic biominerals within the chitin ECM. It has been reported that

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the "bottom-up" method, involving assembling building blocks (cell encapsulating

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microscale hydrogels) into larger tissue constructs,31-34 is an effective strategy to

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obtain a scaffolding material. This method could afford sufficient interconnected 3D

18-20

and the chitinas an original component of

4 ACS Paragon Plus Environment

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pore (channel among the microspheres) structure for rapid cell migration and easily

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adjust to the irregularly shaped bone defects.33-35Thus, we attempted to utilize the

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chitin nanofibrous microspheres with appropriate stabilization capacity, yet without

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any additives, to construct organic-inorganic hybrid microspheres for bone scaffolding.

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It is not hard to imagine that the hierarchicalmicrospheres constructed from the

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biocompatible chitin and HA would be a perfect candidate as a bone scaffold for in

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situ regrowth and regeneration. Our biomaterial design is based on the three features

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as follows. Firstly, the nanofibersconsisted of the stiff chitin chainswith the contour

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length of several micrometers36can act as the nucleus for the growth of HA crystals

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with different morphology. Secondly, the chitin nanofibers are strong and stable37 and

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HA can be tightly embedded in the chitin matrix. Finally, both chitin and HA have

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excellent biocompatibility and biodegradability,8, 36, 38 so their composite materials

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should be biocompatible and biodegradable.

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Herein, the HA with tunable microstructure morphology was coated on the

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nanofibrous chitin microspheres, coded as NCMH, to obtain a bone scaffolding

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materials.

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matrix(ECM)-mimicking nanofibrous hierarchical architecture with controlled HA

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microstructure coating advantageously enhanced the targeted bone cell-material

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interaction, and multi scale channels/pores in the microspheres could promote cell

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invasion and mass transport conditions. As a result, the NCMH bone scaffolding

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materials induced efficiently the bone regeneration at a cell- and factor-free condition,

We

would

proposal

a

hypothesis


that

the

extracellular

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and the homing of the residual cells, specific peptides or growth factors use usually in

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the comparative studies39-40 for the degradable biomaterials to reach a satisfying

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regeneration. Therfore, this work would provide a new avenue to construct

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organic-inorganic hybrid microspheres derived from the natural resource for the bone

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regeneration.

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Experimental Part

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Materials

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Chitin powder was purchased from Golden-Shell Biochemical Co. Ltd.

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(Zhejiang, China). The chitin powder was purified with NaOH, HCl and NaClO2 by a

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procedure described previously and the degree of acetylation of the purified chitin

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was 90 %. The weight-average molecular weight (Mw) was determined to be 53.4 ×

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105 in 5% (w/v) LiCl–DMAc by dynamic light scattering (DLS, ALV/GGS-8F, ALV,

124

Germany). All of the chemical reagents were purchased from commercial sources in

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China, and were of analytical-grade.

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Fabrication of nanofibrous chitin microspheres (NCM)

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The nanofibrous chitin microspheres were prepared as previous report method.36

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7 g of purified chitin powder was dispersed into a 93 g mixture of NaOH, urea, and

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distilled water (11:4:85) by weight with stirring to obtain a suspension. Subsequently,

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the suspension was frozen at -30 oC for 4 h, and then thawed at room temperature.

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The freezing/thawing cycle was repeated twice to obtain a transparent chitin solution, 6 ACS Paragon Plus Environment

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with chitin concentration of 7 wt%. The chitin solution was degassed by

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centrifugation at 7200 rpm for 15 min at 0oC. A well-mixed suspension containing

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50g of isooctane and 1.1 g of Span 85 were dispersed in a reactor. The resulting

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suspension was stirred at 1000 rpm for 30 min, and then 20 g chitin solution was

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dropped into the suspension within 5 min. The suspension was kept stirring for 1h at

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the same stirring speed at 0oC. A solution containing 0.6 g of Tween 85 in5 g

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isooctane was then added to the emulsion and stirred at the same stirring speed for

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another 1 h to achieve stable water/oil emulsion droplets. Subsequently, a 60 oC water

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bath was added to the emulsion for 5 min for the chitin microspheres regeneration. To

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the resultant suspension, dilute hydrochloric acid (10%) was added until the pH

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reached 7. After removing the isooctane, the regenerated nanofibrous chitin

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microspheres with the diameter of 20-120 m in the substratum were obtained. The

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microspheres were washed with deionized water, and then with ethanol successively

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for three times to remove the residual isooctane, Tween 85 and Span 85. Finally, the

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microspheres were subjected to solvent-exchange with t-BuOH. Chitin microspheres

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containing t-BuOH were frozen by immersing into liquid nitrogen, and subjected to a

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conventional freezer dryer.

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Calcium phosphate mineralization on NCM

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The precipitation of calcium carbonate or calcium phosphate was performed

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using the fresh ammonia aqueous solution (30% w/w, 7 mL) or solid ammonium

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carbonate diffusion method in a bell jar at room temperature (20±1℃). The 7 ACS Paragon Plus Environment

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freeze-dried NCM were soaked in precursors aqueous solution (the concentration was

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shown in Table 1)for 12h. Subsequently, the suspension was filtered and the resulted

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swollen NCM was transferred into a bell jar. The ammonia aqueous solution (7 mL)

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or solid ammonium carbonate (5g) was added to a dish and then also placed in the bell

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jar. Addition of different concentrations of CaCl2 was studied (10, 50, and 100 mM).

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CaCl2/NaH2PO4 solution with a calcium-to-phosphate (Ca/P) molar ratio of 1.67,

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consistent with the formation of HA with a formula of Ca10(PO4)6(OH)2, the pH was

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then adjusted to 2.0. However, as these variations only affected the amount of

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precipitated materials and not their nature or structure, the concentration of 100mM

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was used. 24h after ammonia or ammonium carbonate introduction, the mineralized

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NCM was washed with deionized water until pH=7. Samples of calcium carbonate

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containing NCM were converted to calcium phosphate by immersion in a 200 mM

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aqueous solution of sodium phosphate (PB) at pH=7 for 24hand then washed with

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deionized water for 3 days. Finally, the samples were subjected to solvent-exchange

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with t-BuOH and frozen by immersing into liquid nitrogen, followed by freeze-dried

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for characterizations.

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Table 1. Concentrations of salt precursors used in HA synthesis on NCM surface. CaCl2

NaH2PO4

NH3·H2O

(NH4)2CO3

200 mM PB

(mM)

( mM)

(mL)

(g)

(pH=7)

NCMH1

100

－

－

5

√

NCMH2

100

60

7

－

－

NCMH3

100

60

－

5

√

Sample

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Biomacromolecules

Characterization

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Fourier-transform infrared (FT-IR) spectra of the samples were recorded on a

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Perkin-Elmer FT-IR spectrometer (model 1600, Perkin–Elmer Co. USA). The tested

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samples were prepared by the KBr-disk method. Scanning electron microscopy (SEM)

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was performed on a FESEM (SEM, SIRION TMP, FEI) by using an accelerating

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voltage of 5 kV. The samples were sputtered with gold before observation. The

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high-resolution transmission electron microscopy (HRTEM) image was taken on a

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JEOL JEM 2010 FEF (UHR) microscope at 200 kV. The imbibed water of materials

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was exchanged to acetone, and then the microspheres were embedded with epoxy

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resin Epon812 (Shanghai Bioscience, Shanghai, China).After that, the embedded

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specimen was sectioned by a Leica Ultra cut-E using a diamond knife to prepare

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approx.70 nm-thick sections. The wide-angle X-ray diffraction (XRD) was carried out

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on a XRD instrument X’Pert Pro (PANalytical) with Cu-K radiation (=0.154 nm).

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The XRD data were collected from 2= 3 to 60o at a scanning rate of 2o/min. X-ray

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photoelectron spectra (XPS) were recorded on a ESCALAB 250Xi (Thermo Fisher)

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X-ray photoelectron spectrometer, using Al Kα radiation as the excitation source.

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Cell Adhesion and Cytotoxicity Assay

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The preosteoblast extracted from the calvaria of Mus musculus MC3T3-E1

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(ATCC, CRL-2592, U.S.A.) were cultured in α-minimal essential medium (α-MEM,

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Gibco, Life Technologies, Carlsbad, U.S.A.) supplemented with 10% fetal bovine

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serum, containing 100 U/mL penicillin and 100 mg/mL streptomycin. The 9 ACS Paragon Plus Environment

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proliferation of MC3T3-E1 cells on different substrates were characterized by using

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3-(4, 5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay after the cells

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were cultured for 1, 2 and 3 days. MC3T3-E1 cells co-cultured with sterilized NCMH

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microspheres (with a final concentration of 3 mg/mL ) at a density of 4104 cells/mL.

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For MTT assay, at each of the designated time points (1, 2 and 3 days), 40 μL of MTT

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(Sigma, U.S.A.) solution with a concentration of 5 mg/mL in phosphate buffered

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saline (PBS) was added to 400 μL of culture medium and incubated for 4 h at 37 °C.

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After the culture medium was removed, the formazan reaction products were

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dissolved in 400 μL of dimethyl sulfoxide (DMSO) for 20 min. The optical density of

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the formazan solution was read using a microflake reader (Thermo, Multiskn Go) at

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490 nm. Dyes (Calcein AM /Propidium Iodide, LIVE/DEAD Cell Viability Kits,

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Invitrogen)were added to the cells for 15 min. A fluorescence microscope(Nikon

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ECLIPSE Ti) was used to image the microspheres seeded with MC3T3-E1 at the

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corresponding excitation wavelength. For SEM investigations, the samples were

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washed with PBS, fixed in 2.5 wt% glutaraldehyde for 24 h. After they were washed

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with PBS for 3times, the chitin microspheres with cell culture were progressively

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dehydrated in ethanol (50 % to 99%), then solvent-exchange with t-BuOHand

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freeze-dried. The samples were sputtered with gold for SEM observation.

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Animal Tests

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Bilateral critically sized defects of New Zealand white rabbits with body weight

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of 2.0−2.5 kg were created in the radius by removing 15 mm of midshaft diaphyseal 10 ACS Paragon Plus Environment

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bone. The microsphere scaffolds were filled into the defects (right leg), and the pure

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defect without any material was set as blank control (left leg). The wounds were

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closed with silk threads in layers. After surgery, the rabbits were returned to their

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cages and allowed to move freely. All rabbits were injected daily with penicillin

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intramuscularly with a dose of 400 000 units each for 1 week. All the wounds healed

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gradually, and the rabbits remained active with no post surgery complications.

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Animals were kept in the Institute of Experimental Animals of Wuhan University, in

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accordance with the institutional guidelines for care and use of laboratory animals.

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Results and Disscusion

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Construction of the HA-Chitin Hybrige Microspheres

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Scheme 1 provides a schematic roadmap to describe the preparation of HA

225

coated chitin nanofibrous microsphere (NCMH) as bone scaffolding materials. The

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NCMH microspheres were fabricated by a bottom-up approach including two steps as

227

follows. First, the chitin chains in the solution dissolved in NaOH/urea aqueous

228

system at low temperature self-aggregated into the nanofibers under thermal inducing,

229

and then nanofibrous microspheres (NCM) were fabricated via water/oil emulsion

230

method.36 The NCM microspheres have been demonstrated to be an excellent 3D

231

scaffold due to their nanofibrous and multiscale porous architecture for benefiting the

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nutrient transport and cell adhesion. Subsequently, the HA was introduced to form the

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calcium and phosphate salts, which were used as a precursor to form the spherical 11 ACS Paragon Plus Environment

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flake (2D, NCMH1), needle (1D, NCMH2)and submicron-particle (0D, NCMH3)

235

crystals. These HA crystals were assembled onto the chitin nanofibers (1D) on the

236

microspheres. The fabrication of the NCM has been proved to be a physical process,

237

so that almost all acetyl amino groups (DA=91%) were maintained, which could serve

238

as anchoring sites for inorganic ions in HA.28 Thus, theCa2+ could be easily absorbed

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and stabilized onto the NCM through strong physical interactions with acetyl amino

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groups of chitin when the peculiar 3D scaffolds were soaked into the aqueous medium

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containing the calcium and phosphate salts. By using the pure NH3 or mixture of NH3

242

and CO2 diffusion, the nanofibrous chitin microspheres could serve as the crystal

243

nucleis for the crystal growth via the precipitating the calcium phosphate or calcium

244

carbonate. Noticeably, the existence of both hydrophilic hydroxyls and acetyl amino

245

group and hydrophobic pyranose rings of chitin resulted in the amphilicity of the

246

materials, which would induce the crystal growth within control size, and avoid the

247

completely cladding to the polymer matrix through the hydrophobic-hydrophilic

248

interfacial interaction, thus maintaining the chitin naofibrous surface with significant

249

biomedical function. Moreover, the distinctive porous nanofibrous structure could

250

also acted as a template for the crystal growth, leading to them being an

251

biomineralization platform for the bone tissue engineering scaffold application.

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Inspired by the hierarchical biominerals of the arthropods shell in nature, where

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the chitin interacts with calcium carbonate at interfacial, we explored to fabricate the

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calcium carbonate with high stability on the nanofibrous chitin microspheres, 12 ACS Paragon Plus Environment

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subsequently transformed them into HA with sodium phosphate. We selected the

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NCM microspheres with mean diameter around 70 m (Figure 1a, b) as the starting

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materials, because this size could supply a median pore with diameter around 10-20

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m among the microsphere accumulation for the cells invasion.34The CaCO3

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precipitated on the NCM surface through the diffusion of the NH3 and CO2 into the

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CaCl2 aqueous soaked NCM. From the energy-dispersive spectrum (EDS) (Figure S1),

261

there were the carbon and oxygen in

262

nitrogen peaks, indicating the much lower intensity of N, compared with carbon and

263

oxygen. AfterCaCO3 deposition, the carbon, oxygen and calcium appeared in the

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EDX spectrum of the CaCO3 coated NCM (Figure S1). The FTIR (Figure S2a)

265

spectra indicated the characteristic bands at 875 and 746 cm-1 corresponding to out

266

and in-plane bending of CO32−, suggesting the formation of CaCO3.17 The

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XRD(Figure S2b) confirmed the crystalline structure of calcite and aragonite. These

268

results revealed the successful CaCO3 coating on the NCM. Interestingly, the CaCO3

269

coated NCM displayed the same spherical shape and very similar nanofibrous

270

structure(no crystals could be found) within NCM, but an increase of the nanofiber

271

mean diameter (from 28 to 38 nm) (Figure1d, S3c). This could be explained that the

272

chitin nanofibers acted as the templates and stabilizer for the extremely small CaCO3

273

nanoparticles to deposit, so the morphology of the composite microspheres changed

274

hardly, compared to the original one. After treated with the sodium phosphate, the

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flower-like structures with a diameter about 1-3μm composed of fine lamellae (Figure

NCM. However, there were no calcium or


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2a,d) emerged on NCM, and the chitin nanofibers penetrated into the HA crystals

277

(Figure S4), indicating that the NCM microspheres acted as the template and nuclei

278

function. Furthermore, different concentration of Ca2+ only affected the amount of the

279

mineralization but not their nature or structure. At a relatively low Ca2+ concentration

280

(50 mM), small flake crystals with diameter about 500 nm (Figure S5a)derived from

281

the limited Ca2+ adhered on the chitin nanofibers, suggesting the crystal nuclei

282

function of the chitin nanofiber. Unexpectedly, when the Ca2+ concentration increased

283

to 150 mM, only small flake crystals with diameter about 400 nm(Figure

284

S5b3)instead of a larger or denser flower HA crystals than that fabricated in 100 mM

285

Ca2+ could be obtained, but flake-like crystal with large size existed outside the NCM.

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This phenomenon arose from the fact that acetyl amino groups on the NCM have a

287

saturated absorbed capacity for Ca2+, which could only bound a certain amount of

288

CaCO3 crystal nucleis due to the amphilicity of the chitin, whereas large amounts of

289

free CaCO3 crystal nuclei resulted from excessively high concentration of Ca2+ had a

290

strong tendency of self-aggregation with each other and adsorbed the Ca2+ and CO32+

291

quickly in aqueous to grow into a large crystal. After treated with sodium phosphate

292

for HA transformation, only small flake HA crystals with diameter about 400 nm

293

were fabricated on NCM, and large size crystal occurred outside the microsphere

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(Figure S5b1).

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To explore to deposit HA directly on NCM, we used a one-pot synthesized

296

crystallization method. During the reaction process, the pure NH3 or NH3 and CO2 14 ACS Paragon Plus Environment

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vapor diffused into a CaCl2-NaH2PO4 mixed solution (Scheme 1). The pure NH3

298

induced the needle-like topography (Figure 2b, e), whereas the NH3 and CO2 led to a

299

submicron-sphere crystal structure (Figure 2c, f) with the mean diameter about 315

300

nm, and they are penetrated by the chitin nanofibers. Both needle and

301

submicron-sphere crystals grew uniformly around the surface of the chitin

302

nanofibrous microsphere, owing to the template and crystal nuclei function of the

303

chitin for mineralization. Due to the small size of the submicron-sphere and

304

submicron-needle HA, the Ca and P element were distributed homogeneously on the

305

chitin microsphere surface(Figure 2). On the contrary, the micro size of the microflake

306

HA crystals made they couldn’t coat on the chitin microsphere uniformly, and the

307

flower-like edge of the Ca and P clearly indicated the boundary between the HA and

308

chitin nanofibrous surface.

309

Structure and Interface Interaction of HA and NCM

310

From the FTIR and XRD results (Figure S6), the characteristic band of CO32+

311

(874 and 747 cm-1) and PO43+(560-660 and 950-1100 cm-1) identified to CaCO3 and

312

HA,41-44 respectively, confirming the formation of CaCO3-HA nanocomposites, as a

313

result of the introduction of both CO2 and NH3 (the CaCO3 induced the sphere

314

structure).45 It was noted that the mean diameter (310 nm) and morphology of the

315

submicron-sphere crystals on NCM(Figure S7)changed hardly after the HA

316

transformation through immersion in the sodium phosphate. The different

317

concentration of Ca2+ and phosphate were also employed for the mineralization on the 15 ACS Paragon Plus Environment

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318

NCM, and they just affected the density and crystal size of the crystal but not their

319

nature and structure. For the needle and sphere-like crystals (Figure 3, 4 and S7, 8),

320

within the Ca2+ concentration below 150 mM and 100 mM. The higher concentration

321

led to a greater density and larger size of the HA coating on the NCM, whereas when

322

it reached up to 200mM and 150mM, respectively, the amounts and mean size of the

323

submicron-needle(21, 24, 32, 39, 21 nm width

324

precursor) and submicron-sphere (255, 310, 175, 163 nm diameter for 75, 100, 150,

325

200 mMCa2+ precursor)HA on the NCM decreased, and even some large flake

326

aggregation of HA crystals appeared on the NCM for the submicron-sphere crystals.

327

This could be explained by the facts that (1) the amphilicity of the chitin led to an

328

appropriate affinity to the inorganic submicron-particles, and the chitin nanofibers

329

could only adsorb certain amount of Ca2+ for the HA crystals growth, as a result the

330

HA crystal size on the NCM could be controlled in submicron-scale size; (2) large

331

amounts of free crystal nucleis existed in aqueous as mentioned above and they would

332

self-aggregated into large size crystals. Interestingly, the sphere-like crystal displayed

333

a ring structure with a pore through. The chitin nanofiber couldn’t be observed by the

334

TEM for its low contrast. Thus, the NCMH hybrid microspheres with different size

335

and

336

submicron-sphere HA crystals, corresponding to NCMH1, NCMH2 and NCMH3,

337

were successfully constructed via the in-situ synthesis method.

shape

such

as

the

large

flake

for 25, 50, 100, 150, 200mMCa2+

(microflake),


submicron-needle

and

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Biomacromolecules

338

To clarify the crystalline structure of the calcium phosphate coating, the

339

microspheres were observed by XRD (Figure 5A). Besides the typical -chitin

340

crystalline structure,46 all the samples displayed a low crystallinity HA architecture,

341

which have been known for its good in vivo absorbability and efficient osteogenesis.15,

342

19

343

chitin microspheres and HA nanoparticles was ascertained using X-ray photoelectron

344

spectroscopy (XPS). The overview spectra (Figure 5B) demonstrated that the C, O,

345

N, Ca and P existed in the NCMH microspheres, while only C, O and N in the NCM.

346

Interestingly, the peak of N in NCMH1,2,3 shifted 0.1, 0.3, 0.4 to a lower binding

347

energy (Figure 5C). This indicated that the N in the acetyl amino groups acted as the

348

anchor for the HA nanoparticles, leading to their immobilization on NCM. The less

349

joints between the flake crystals and chitin nanofibers arising from the larger size led

350

to the smaller N shift of NCMH1. In view of these above results, the NCMH

351

hybridmicrospheres were constructed by coating HA on the chitin nanofibers, which

352

penetrated into hydroxyapatite crystals, leading to the hierarchical structure.The

353

acetyl amine and hydroxyl groups on chitin could interact with the HA so that the

354

chitin nanofibers could anchor the HA crystals through the non covalent bond (Figure

355

5d). The relatively hydrophobic pyranose rings of chitin avoid the excessively large

356

size HA growth within NCM matrix. Thus, the obtained HA crystals could be

357

controlled in a submicron-scale size and the nanofibrous surface of the NCM could be

358

maintained after the in-situ mineralization.Furthermore, the hydroxyl groups on the

More detailed information regarding the chemical and bonding environment of the


Biomacromolecules

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

359

chitin may perform the hydrogen bonding to bound and stabilize the HA crystals

360

during the mineralization process. Therefore, the tight interface adhesionbetween

361

chitin and HAcrystals through the non covanlent bonds existed in the hybrid

362

microspheres,

363

Inducing Bone Regeneration

364

It was demonstrated that the cell functions such as cell adhesion, migration and

365

proliferation respond differently to substrates with varied microscopic architecture.

366

The surface topography and roughness of calcium phosphate or their composition

367

with polymers have been reported to possess significant effects on human bone cells,

368

especially on their proliferation and differentiation.47Thus, the NCMH microspheres

369

with different HA morphology were further evaluated on their biofunction as bone

370

scaffolding. We hypothesized that the HA coating morphology on the chitin

371

microsphere was highly relevant to guide cell adhesion and proliferation on the

372

NCMH microsphere scaffold, which would affect the bone scaffolding performance

373

of the microsphere bone grafting. Moreover, such surface with microstructure,

374

together with the interconnected pore resulted from the cavities among the

375

microsphere accumulation (Figure S9b) could benefit to the potential bone scaffolding

376

performance. We examined the chitin–HA microspheres as a bottom-up building

377

block scaffolding for bone regeneration using two experimental models: (1) in vitro

378

osteoblast precursor cell line culture; (2) rabbit radius defect repair. The in vitro cell

379

proliferation of MC3T3-E1 cells (an osteoblast precursor cell line for bone) on the 18 ACS Paragon Plus Environment

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Biomacromolecules

380

NCMH microspheres with different HA structure coatings was examined and

381

compared with that of the pure chitin microspheres. After 3 days of culture, the cell

382

viability on the NCMH microspheres increased obviously, compared to the pure chitin

383

microspheres (Figure S9a), indicating that the HA coatings enhanced the

384

biocompatibility of the hybrid chitin microspheres for the osteoblast cell. Interestingly,

385

though the similar crystalline structure and physicochemical property of the HA

386

coatings, the cells number on submicron-needle and submicron-sphere HA coating

387

microspheres were significantly higher than that of the micro-flake HA-chitin

388

microsphere. The enhanced osteoblast proliferation was further confirmed by the

389

florescent photographs of cells cultured on the NCMH microspheres (Figure 6a-h).

390

The cells proliferated on all the microspheres, and the proliferated cell number were

391

significantly higher on the submicron-needle and submicron-sphere HA coating

392

microsphere than that of the micro-flake，and this was consistent with the MTT results.

393

It could be concluded that the addition of the HA crystals significantly improved the

394

cytocompatibility of the NCMH microspheres as well as the osteoblast adhesion and

395

proliferation. From the SEM images (Figure 6i–p), a clearer insight of the interaction

396

between cells and microsphere appeared. The preosteoblast cells filopodium extended

397

and adhered on the surface of the peripheral microspheres (Figure 6i-l). The cells only

398

attached the single nanofibrous surface or micro-flake HA on chitin and NCMH1

399

microspheres (Figure 6m, n and S9c, d). On the contrary, both the submicron-fibrous

400

surface and submicron-scale HA crystals of the NCMH2 and NCMH3 could be “felt” 19 ACS Paragon Plus Environment

Biomacromolecules

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

401

by the preosteoblast cells (Figure 6o, p and S9e, f). The high cell proliferation ratio of

402

NCMH2 and NCMH3 could be attributed to the homogeneous submicron-scale HA

403

crystals on the nanofibrous surface, which make the cells could attach to the chitin

404

nanofibers and the HA submicron-crystals and their biofunctions of promoting cell

405

adhesion and osteoconduction could be both maintained (Figure S9e, f). However, the

406

micro-flake HA covered largely on the chitin nanofibrous surface to hinder the contact

407

between the nanofibrous and the cells (Figure S9d), leading to a relatively low cell

408

proliferation. In view of the results above, the HA submicron-crystals coated scaffold

409

provided a suitable physicochemical and biological microenvironment for the

410

osteoblast precursor cells to attach, which is an important factor in determining the

411

eventual result of in vivo tissue regeneration. As a result, the NCMH2 and NCMH3

412

(better in vitro cell proliferation performance) were selected for further in vivo

413

cell-free bone repair analyses. It is well known that HA has been widely adopted as

414

the artificial bone in clinical bone defect therapy for many years, thus we used them

415

as a standard by which the performance of our NCMH were judged.

416

The widely used radius defect model was selected as the standard to evaluate the

417

bone regeneration performance of the NCMH.48 In a typical rat radius defect model, a

418

critical length of 15 mm bone defect was created (Figure 7b). A fracture defect of this

419

size cannot be healed during the lifetime of the animal.49 The as-prepared scaffold

420

with cell-free seeded were implanted into the critical size (Figure 7c). The NCMH

421

microsphere could well adjust the irregular bone defect shape. More importantly, the 20 ACS Paragon Plus Environment

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Biomacromolecules

422

interconnected channel among the microspheres could allow the fast cells invasion

423

and proliferation on the all microsphere scaffold (Figure 7a). After twelve weeks of

424

implantation, the digital radiographs (Figure 7d) and hematoxylin and eosin (HE)

425

staining images (Figure 8) showed that, except for the blank group (the left), all the

426

defects implanted with the scaffolds were bridged by the newly formed bone(Figure

427

8). However, the defect area in the control group was primarily filled up with fibrous

428

soft tissue and without marked bone formation (HE in Figure 8), indicating a reliable

429

animal model. Interestingly, the NCMH2 and NCMH3 microspheres exhibited the

430

better bone fusion with the clear outlines of cortex and larger amount of new bone

431

(NB) than that of the pure chitin microsphere or HA submicron-crystals (Figure 7d).

432

More surprisingly, even some obvious bone marrow cavities and vessel structure

433

occurred in the NCMH2 and NCMH3, suggesting that the regenerated bone have a

434

relatively

435

submicron-crystals coating microsphere successfully combined the both biofunctions

436

of HA and chitin nanofibrous microspheres, leading to the significantly promoting

437

bone defect healing. With the new bone (NB) formed, the NCMH2 and NCMH3 were

438

adsorbed gradually, and the unhealed defect area was still filled with the microspheres

439

during the healing process (data not shown). After 12 weeks, when the defect was

440

almost entirely regenerated, only 12 % (NCMH2) and 11 % (NCMH3) microspheres

441

were left, and no microspheres were observed after 16 weeks. These results indicated

442

the biodegradability of NCMH in vivo. These findings demonstrated that a

perfect

bone

structure.

These

results


indicated

that

the

HA

Biomacromolecules

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

443

microsphere scaffold with appropriate surface properties together with dynamic

444

biodegradation can guide the in vivo bone regeneration under clinically challenging

445

healing conditions to relieve the pain of patients.

446 447

Conclusion

448

The nanofibrous chitin microspheres (NCM) were fabricated from the chitin

449

solution in NaOH/urea aqueous system with cooling, and subsequently the NCMH

450

hybrid microspheres coated with different size and shape HA submicron-crystals

451

including the micro-flake, submicron-needle and submicron-sphere were constructed

452

successfully through the non covanlent bonds, generating a new class of the

453

bottom-up bone grafting scaffold. In the NCMHmicrospheres, the chitin nanofibers

454

penetrated into hydroxyapatite crystals, leading to the hierarchical structure. The

455

introduced HA submicron-crystals effectively enhanced the osteoblast precursor cell

456

proliferation on the NCMH microspheres. The hybrid microspheres consisted of

457

needle and/or sphere HA submicron-crystals and the chitin nanofibers exhibited

458

excellent biofunctions for promoting the cell adhesion and the high inducing

459

osteoconduction, as a result of the combinationthe inherent biocompatibilitiesand the

460

hierarchical microstructure ofboth the chitin nanofibrous and HA. The NCMH

461

microspheres could effectively heal the bone defect in vivo without external

462

preloading with cells or simultaneous application of bioactive growth factors.

463

Therefore, the NCMH microspheres could inducewell the bone regeneration, and 22 ACS Paragon Plus Environment

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Biomacromolecules

464

provided the sufficient channels for the cells invasion and proliferation, as well as the

465

flexible adaption for the irregular shape defects. This new appealing concept for bone

466

scaffold discovered in this study could serve as a design consideration for

467

next-generation bone grafts in tissue engineering.

468

.

469

Acknowledgements

470

This work was supported by the Major Program of National Natural Science

471

Foundation of China (21334005), theMajor International (Regional) Joint Research

472

Project of National Natural Science Foundation of China (21620102004), and the

473

National Natural Science Foundation of China (20874079).

474 475 476

ASSOCIATED CONTENT.

477

Supporting Information.

478

EDX, XRD and FTIR profiles of the NCM, CaCO3 coated NCM and

479

submicron-sphere crystal coated NCM. SEM images of the CaCO3 coated NCM and

480

microflake calcium phosphate with different Ca2+ concentration coated NCM. SEM

481

images of the submicron-sphere size and its distribution. The width distribution of the

482

HA submicron-needle crystals. MTT assay for the cell viability on native and calcium

483

phosphate coated naofibrous chitin microspheres. The description of the contact of the

484

cells to the hybrid microspheres. This information is available free of charge via the

485

Internet at http://pubs.acs.org/ 23 ACS Paragon Plus Environment

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486 487

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Wang, L.; Zhou, G.; Liu, H.; Niu, X.; Han, J.; Zheng, L.; Fan, Y., Nanoscale

Zhang, P.; Hong, Z.; Yu, T.; Chen, X.; Jing, X., Biomaterials 2009,30,

Mokbel, N.; Bou Serhal, C.; Matni, G.; Naaman, N., Oral and Maxillofacial

585 586


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Biomacromolecules

587

Figure captions

588

Scheme 1. Schematic formation process of microflake (NCMH1), submicron-needle

589

(NCMH2) and submicron-sphere(NCMH3) hydroxyapatite crystals coated on the

590

nanofibrous chitin microspheres

591

Figure 1. SEM images of nanofibrous chitin microspheres (a, b, c) and the diameter

592

distribution (d) of chitin nanofibers.

593

Figure 2. SEM images of the NCMH1 (a, d), NCMH2 (b, e) and NCMH3 (c, f);

594

Element mapping of Ca and P ford, e ,f; TEM images of the NCMH1 (g), NCMH2 (h)

595

and NCMH3 (i).

596

Figure 3.SEM images of the submicron-needleHA deposited on NCM with

597

concentration of Ca2+ precursor solution for 25 (a, e), 50 (b, f), 150 (c, g) and 200 mM

598

(d, h); the width distribution (i, j, k, l) of the HA submicron-needle crystals in (e, f, g,

599

h).

600

Figure 4.SEM images of the submicron-sphere HA deposited on NCM with

601

concentration of Ca2+ precursor solution for 75 (a, d), 150 (b, e) and 200 mM (c f); the

602

diameter distribution (g, h, i) of the HA submicron-sphere crystals in (d, e, f).

603

Figure 5. XRD spectra (A), XPS fully scanned spectra (B) and XPS spectra of N1s

604

(C) for NCMH1 (a), NCMH2 (b), NCMH3 (c) and NCM (d); some dicalcium

605

phosphate dehydrate (DCPD) existed in a (A); the hydroxyl and acetyl amino groups

606

om chitin nanofibers interacted with the HA crystals throug the non covalent bond.


Biomacromolecules

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

607

Figure 6. Bright field (a-d), LIVE assay fluorescent image (e-h) and SEM images of

608

MC3T3-E1 cells cultured on NCM (i, m), NCMH1 (j, n), NCMH2 (k, o) and NCMH3

609

(l ,p). The scale bar are (a-d) 20m, (e-h) 20 m, (i-l) 10 m and (m-p) 2 m.

610

Figure 7. Strategy for HA coated chitin nanofibrous microspheres induced bone

611

regeneration (a); the rabbit radius bone defect (b) and microsphere as bone grafting

612

implanted (c); X-ray examination (d) of the original bone defects and the defects

613

treated with NCM, HA, NCMH2 and NCMH3 for 12 weeks.

614

Figure 8. The photograph and HE staining images (e) of the regeneration bone in d;

615

More new bone (NB), vessel (green circle, V) and bone marrow (blue circle, BM) are

616

marked in e; The scale bar are (b, c, d, photograph in e) 1 cm, (HE 40×) 50 m and

617

(200×) 10 m.

618 619


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Biomacromolecules

620 621

Scheme 1. Schematic formation process of microflake (NCMH1), submicron-needle

622

(NCMH2) and submicron-sphere(NCMH3) hydroxyapatite crystals coated on the

623

nanofibrous chitin microspheres

624 625 626 627 628


Biomacromolecules

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

629 630 631 632 633 634 635 636 637 638 639 640 641 642 643

Figure 1. SEM images of nanofibrous chitin microspheres (a, b, c) and the diameter

644

distribution (d) of chitin nanofibers.

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Biomacromolecules

646 647

Figure 2. SEM images of the NCMH1 (a, d), NCMH2 (b, e) and NCMH3 (c, f);

648

Element mapping of Ca and P ford, e ,f; TEM images of the NCMH1 (g), NCMH2 (h)

649

and NCMH3 (i).The Ca2+ precursor concentration = 100 mM.

650


Biomacromolecules

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

651 652

Figure 3.SEM images of the submicron-needleHA deposited on NCM with

653

concentration of Ca2+ precursor solution for 25 (a, e), 50 (b, f), 150 (c, g) and 200 mM

654

(d, h); the width distribution (i, j, k, l) of the HA submicron-needle crystals in (e, f, g,

655

h).

656 657


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Biomacromolecules

658 659

Figure 4.SEM images of the submicron-sphere HA deposited on NCM with

660

concentration of Ca2+ precursor solution for 75 (a, d), 150 (b, e) and 200 mM (c f); the

661

diameter distribution (g, h, i) of the HA submicron-sphere crystals in (d, e, f).

662 663 664 665 666 667 668 669 35 ACS Paragon Plus Environment

Biomacromolecules

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

670 671

Figure 5. XRD spectra (A), XPS fully scanned spectra (B) and XPS spectra of N1s

672

(C) for NCMH1 (a), NCMH2 (b), NCMH3 (c) and NCM (d); some dicalcium

673

phosphate dehydrate (DCPD) existed in a (A); the hydroxyl and acetyl amino groups

674

om chitin nanofibers interacted with the HA crystals throug the non covalent bond.

675


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Biomacromolecules

676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692

Figure 6. Bright field (a-d), LIVE assay fluorescent image (e-h) and SEM images of

693

MC3T3-E1 cells cultured on NCM (i, m), NCMH1 (j, n), NCMH2 (k, o) and NCMH3

694

(l ,p). The scale bar are (a-d) 20m, (e-h) 20 m, (i-l) 10 m and (m-p) 2 m.

695 696


Biomacromolecules

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

697 698

699 700

Figure 7. Strategy for HA coated chitin nanofibrous microspheres induced bone

701

regeneration (a); the rabbit radius bone defect (b) and microsphere as bone grafting

702

implanted (c); X-ray examination (d) of the original bone defects and the defects

703

treated with NCM, HA, NCMH2 and NCMH3 for 12 weeks.

704 705


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Biomacromolecules

706 707

Figure 8. The photograph and HE staining images (e) of the regeneration bone in d;

708

More new bone (NB), vessel (green circle, V) and bone marrow (blue circle, BM) are

709

marked in e; The scale bar are (b, c, d, photograph in e) 1 cm, (HE 40×) 50 m and

710

(200×) 10 m.

711 712


Biomacromolecules

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

713

Hierarchical Microspheres Constructed fromChitin

714

Nanofibers Penetrated Hydroxyapatite Crystalsfor Bone

715

Regeneration

716 717 718

Bo Duana, Kangquan Shoub, Xiaojuan Sua, Yahui Niub, Guan Zhengc, Yao Huanga,

719

Aixi Yub, Yu Zhangc, Hong Xiac, Lina Zhanga

720 721

a

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072,

722

China b

723 724 725

c

Zhongnan Hospital, Wuhan University, Wuhan 430072, China

Department of Orthopedics, General Hospital of Guangzhou Military Area Command of Chinese PLA, Guangzhou 510010, China

726

* To whom correspondence should be addressed. Phone: +86-27-87219274. Fax:+86-27-68754067.E-mail:,[email protected],(L. Zhang) 40 ACS Paragon Plus Environment

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Hierarchical Microspheres Constructed from Chitin Nanofibers

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