Lightweight Open-Cell Scaffolds from Sea Urchin Spines with Superior

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Lightweight open-cell scaffolds from sea urchin spines with superior material properties for bone defect repair Lei Cao, Xiaokang Li, Xiaoshu Zhou, Yong Li, Kenneth Scott Vecchio, Lina Yang, Wei Cui, Rui Yang, Yue Zhu, Zheng Guo, and Xing Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01645 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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ACS Applied Materials & Interfaces

Lightweight open-cell scaffolds from sea urchin spines with superior material properties for bone defect repair Lei Cao†,# , Xiaokang Li‡,#, Xiaoshu Zhou¶, Yong Li‡, Kenneth S. Vecchio , Lina Yang†, Wei Cui†, Rui Yang†,§, Yue Zhu¶,*, Zheng Guo‡,*, Xing Zhang†,§,* ￥

†

Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning 110016, China ‡ Department of Orthopedics, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China ¶ Department of Orthopedics, The First Hospital of China Medical University, Shenyang, Liaoning 110001, China ￥ Nanoengineering Department, University of California, San Diego, La Jolla, CA 92093, USA § University of Science and Technology of China, School of Materials Science, Hefei, Anhui, 230026, China

ABSTRACT: Sea urchin spines (Heterocentrotus mammillatus), with hierarchical open-cell structures similar to human trabecular bone, and superior mechanical property (compressive strength ~43.4 MPa) suitable for machining to shape, are explored for potential applications to bone defect repair. Finite element analyses reveal that the compressive stress concentrates along the dense growth rings and dissipates through strut structures of the stereoms, indicating that the exquisite mesostructures play an important role in high strength-to-weight ratios. The fracture strength of magnesium-substituted tricalcium phosphate (β-TCMP) scaffolds, produced by hydrothermal conversion of urchin spines, is about 9.3 MPa, comparable to that of human trabecular bone. New bone forms along outer surfaces of β-TCMP scaffolds after implantation in rabbit femoral defects for one month, and grows into the majority of the inner open-cell spaces post-operation in three months, showing tight interface between the scaffold and regenerative bone tissue. Fusion of beagle lumbar facet joints using a Ti-6Al-4V cage and β-TCMP scaffold can be completed within seven months with obvious biodegradation of the β-TCMP scaffold, which is nearly completely degraded and replaced by newly-formed bone after ten months implantation. Thus, sea urchin spines suitable for machining to shape have advantages for production of biodegradable artificial grafts for bone defect repair. KEYWORDS: sea urchin spines; lightweight scaffolds; open-cell structure; mechanical property; finite element analysis; bone defect repair

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1. INTRODUCTION In recent decades, cellular materials including closed-cell materials (alternatively called honeycomb-like materials, such as wood, cork, etc.) and open-cell materials (alternatively called foam-like materials, such as metallic foams, marine sponges, coral, etc.) ,1 with good physical,2-4 mechanical,5 and thermal properties,6 have been of great interest for a variety of applications in the fields of aerospace,7 architecture,8 chemical engineering,9 biomedical engineering,10 among others.11 Compared to closed-cell materials, open-cell materials comprising trabecular-like mesh structures generally show higher strength-to-weight ratios. Moreover, these open-cell materials have advantages for particular applications, such as bone graft substitutes, considering that open-cell structures allow essential cell infiltration and nutrient transportation like trabecular bone.12 Porous calcium phosphate bioceramics including hydroxyapatite (HAP) and β-tricalcium phosphate (β-TCP) have been widely used as scaffolds for bone defect repair due to their good biocompatibility and osteoconductivity.13 Macroporous calcium phosphate ceramics (i.e. pore size > 100 µm), with high porosity and interconnected porous structures, are preferred for tissue ingrowth. They can serve as excellent temporary scaffolds for cell interactions, extracellular matrix deposition, and blood vessel ingrowth, thus providing essential structural support to the newly-formed bony tissue.14 Many porous calcium phosphate bioceramics fabricated using porogen agents or polymeric templates mainly exhibit closed-cell structures,15 which have relatively poor mechanical strength at high porosity, potentially leading to corruption during implantation and migration of the resulting ceramic debris to adjacent soft tissues. Moreover, machinability, to shape these brittle porous scaffolds is extremely difficult, restricting their use as small bone defect repair. Therefore, scaffolds that have open-cell structures and superior mechanical strength suitable for machining to shape, are particularly preferred for bone defect repair in such cases. Biological materials including sea urchin spines,16 cuttlebone,17,18 and marine


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spongies19 created by mother nature have hierarchical open-cell structures, which can be used to produce scaffolds for bone defect repair. Previous studies reveal that spines of sea urchin, e.g. Heterocentrotus mammillatus and Heterocentrotus trigonarius, consist of magnesian calcite mesocrystals formed through a phase-transition from initially amorphous CaCO3 to crystalline, and show concentric-ring corticle-trabecular mesostructures.20-23 Sea urchin spines can be converted to magnesium-substituted tricalcium phosphate (β-TCMP) scaffolds using a hydrothermal reaction, while maintaining their original open-cell structures.16,18 The resulting β-TCMP scaffolds show good biocompatibility and form a tight bond to regenerative bone tissue in vivo using the rat femoral bone defect model.16 However, bone ingrowth into porous structures and degradability of these β-TCMP scaffolds in vivo, have not been investigated to date. Moreover, the underlying relationship between mechanical properties and the hierarchical structures of sea urchin spines is not well understood, which is of great interest when using these materials as bone graft substitutes. In this study, the relationship between mechanical property and open-cell structures of sea urchin spines is investigated using experimental mechanical testing coupled with finite element analysis (FEA). The interaction of open-cell structures of the resulting β-TCMP scaffolds and the regenerative bone tissue, including tissue ingrowth and the scaffold degradability, is examined by implantation of the scaffolds in vivo using a rabbit femoral defect model and a beagle lumbar facet joint fixation model. 2. EXPERIMENTAL SECTION 2.1. Sample preparation. Sea urchin (Heterocentrotus mammillatus) spines with a thickness of ~1 cm and lengths of ~8.5-11.5 cm were bought from a local sea product store, which were cut into particular sizes for further experiments. Sea urchin spine samples were immersed into a NaClO solution (10 wt.%) for 30 min and subsequently boiled in deionized water for an hour to remove organic components. These samples were further rinsed with deionized water and then reacted with a 0.1 g/ml



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(NH4)2HPO4 solution at 200°C for two days in a hydrothermal reaction vessel to produce magnesium substituted tri-calcium phosphate (β-TCMP) scaffolds following the ion-exchange reaction (equation (1)): 3(Ca,Mg)CO3+2(NH4)2HPO4→(Ca,Mg)3(PO4)2+2(NH4)2CO3+H2CO3

(1)

All chemicals at analytical grade were bought from Sinopharm Chemical Reagent, Shanghai, China. 2.2. Composition and crystal phase analysis. The sample crystal phases were identified by powder X-ray diffraction (XRD), which was operated at 40 kV and 100 mA at a 2θ range of 20°-70° with a 0.02° step size using a Rigaku D/max 2400 diffractometer (Rigaku Corporation, Tokyo, Japan) with monochromated Cu Kα radiation (Kα1 = 1.5418 Å). Sea urchin spine samples before and after the hydrothermal reaction were ground into homogeneous powders for XRD tests to obtain crystal phase information of bulk minerals. Attenuated total reflectance (ATR) of the above powders was performed using a Fourier transform infrared spectrometer (FTIR, TENSOR27, Bruker, Germany) to identify the functional groups in the samples, with the wavenumber from 500 cm-1 to 3500 cm-1 and a step size of 2 cm-1. Calcium and magnesium contents in sea urchin spines were analyzed using the inductively coupled plasma mass spectrometry (ICP-MS, Prodigy, Leeman Labs, Hudson, USA). The samples were dissolved in a 2 wt.% nitric acid solution for ICP-MS tests, and three replicates were conducted. 2.3. Scanning electron microscopy (SEM). The microstructures for sea urchin spine samples before and after the hydrothermal reaction, samples soaked in simulated body fluid (SBF) and implants for rabbit femoral defect repair and beagle lumbar facet joint fixation, were observed with a LEO Supra 35 field emission scanning electron microscope (Zeiss, Jena, Germany) at an accelerating voltage of 20 kV. The samples were coated with a gold film using the sputter coating instrument (Cressington, Watford, England) prior to SEM observation.


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2.4. Micro-CT analysis. The implant samples for rabbit femoral defect repair were scanned with Micron X-ray 3D Imaging System (Cheetah, Germany) to determine the new bone formation. The X-ray source voltage was 90 kV, and the beam current was 50.0 µA. The scanning resolution is ~17 µm. The area of the implant was selected as the region of interest (ROI). The 450 projections were reconstructed using a modified parallel Feldkamp algorithm, and segmented into binary images (12-bit TIF images). The stereoscopic structures of sea urchin spines and implants for beagle lumbar facet joint fixation were characterized by the three-dimensional (3D) high-resolution transmission X-ray tomography (HRTXRT) technique using the Xradia Versa XRM-500 system (ZIESS, Jena, Germany), with the accelerating voltage of 140 kV. A total of 2000 images, each exposed for four seconds, were recorded as the sample was rotated for 360°, which were then computationally reconstructed via a filtered back projection algorithm to produce a 3D image with a voxel size of ~3.5 µm. The 1000 projections were reconstructed using a modified parallel Feldkamp algorithm, and segmented into binary images (8-bit TIF images). The area of the initial β-TCMP implant was selected as ROI. The percentages of the bone volume out of ROI and the remaining β-TCMP scaffold out of ROI were calculated using the threshold of 120-210 for bone and the threshold of 210-255 for the scaffold. The results were presented as the means ± standard deviation (S.D.) for each group. 2.5. Uniaxial compression tests and finite element analysis. Sea urchin spines were cut into cylindrical samples (φ10 mm × 13 mm). Uniaxial compression tests for samples before and after hydrothermal conversion were performed at a constant strain rate of 1×10-3 s-1 at room temperature in air using an Instron 8850 (Instron, Grove, USA). The finite element analysis (FEA) under the small-scale yielding condition was performed using ANSYS software (ANSYS, Pittsburgh, USA) from National Supercomputing Center in Shenzhen for evaluation of stress and strain distribution in original sea urchin spine samples under the quasi-static compression loading. Solid 185-type elements (8 nodes solid structure) were chosen as a unit for the FEA model. The total number of nodes was 258,862, and the total number of elements was



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1,232,105 for one individual FEA model of a sample size (340µm × 340µm × 170µm). Young’s modulus E = 7.61×104 MPa and the Poisson’s ratio γ = 0.3 were used for sea urchin spines.24,25 2.6. In vitro bone mineral formation using SBF. Simulated body fluid (SBF) was considered as an efficient method to evaluate the bone integration ability of implants in body environment.26 In this experiment, SBF buffered at pH = 7.40 with a Tris-HCl solution was prepared as previously reported.27 Converted sea urchin spine samples (β-TCMP scaffolds) were soaked in SBF at 37oC, which was renewed every two days. The specimens were removed from SBF after soaking for 1, 2, and 3 weeks, which were then rinsed with deionized water and dried in an oven at 100oC. The formation of bone mineral-like apatite layers on the surfaces of the implants were observed by SEM. 2.7. Rabbit femoral defect repair. The cylindrical samples (5 mm (diameter) x 10 mm (length)) with drilled holes (diameter ~500 µm)) were converted to β-TCMP scaffolds by a hydrothermal reaction at 200°C for two days (Figure. 1g), which were then used as implants for rabbit femoral defect repair to study bone integration and further growth into the porous structures. New Zealand white rabbits (male) with an average weight of ~2.5-3.5 kg were anesthetized with phenobarbital sodium via intravenous (IV) injection. The surgical area was shaved and sterilized, and an incision about 2 cm long was created to expose the lateral femoral epicondyle. Cylindrical defects (~5 mm (diameter) x 10 mm (length)) were then drilled on the lateral femoral epicondyle, which were filled with β-TCMP scaffolds (n = 9) or left empty as the control group (n = 9). The incisions were then closed with sutures and the surgical area was sterilized again. To prevent wound infection, each animal was given 40000 U of penicillin per day via intramuscular injection for three days after surgery. After implantation for 1, 2, and 3 months, three rabbits were sacrificed by IV injection of air under anesthesia at each time points. The femora condyles samples were harvested and fixed immediately by 4% paraformaldehyde in PBS for seven


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days, which were further used for micro-CT analysis. For histological analysis, the samples were dehydrated in graded ethanol solutions from 70% to 100% and finally embedded in methyl methacrylate (MMA). The embedded samples were sliced (about 50 µm in thickness) using an interlocked diamond saw (Leica Microtome, Wetzlar, Germany) and stained with 1.2% trinitrophenol and 1% acid fuchsin (Van-Gieson staining). All experimental procedures were approved by the ethics committee of the Fourth Military Medical University, according to the relevant guidelines and regulations. 2.8. Beagle lumbar facet joint fixation. The urchin spine samples were machined into an ellipse shape to fit into Ti-6Al-4V fusion cages (Dingjian Medical Device Company, Changzhou, China), and eleven holes (diameter ~500 um) were drilled through the urchin spine samples for better bone ingrowth in vivo. The β-TCMP scaffolds after hydrothermal conversion of urchin spines were positioned in the Ti-6Al-4V fusion cages (Figure. 1h), which were further used for fixation of lumbar facet joint of beagles (weight ~9.8-10.5 kg). Each animal was sedated with IV injection of ketamine (10 mg/kg) and diazepam (0.25 mg/kg) anesthetic medications, followed by endotracheal intubation and general anesthesia using 1.5-2.0% isoflurane. With the animal positioned prone, the posterior lumbar region was shaved, aseptically prepared and draped in sterile fashion. Prophylactic antibiotics (with 1 g cefazolin sodium) were IV administered pre- and post-operatively. A localization radiograph was obtained before surgical intervention of lumbar facet joints of a beagle to ensure visualization of the L3–L4 and L5–L6 vertebral levels. The laminae facet joints and transverse processes were exposed through a midline incision. The posterior capsules of the facet joints were excised, and the articular cartilages were denuded. Ti-6Al-4V fusion cages with β-TCMP scaffolds (n = 8) or autogenous bone (n = 8) were bilaterally grafted into the facet joints at the L3–L4 and L5–L6 levels, which were implanted for 7 or 10 months (n = 4 per sample for each time point). The beagles were sacrificed by IV injection of air under anesthesia, and the implants with adjacent bone were harvested after implantation for 7 or 10 months. The implant samples were fixed



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by 4% paraformaldehyde in PBS for seven days, which were further used for micro-CT analysis and sectioned for SEM analysis. All animal surgeries and experimental procedures commenced after protocol approval by the Animal Ethics Committee of the China Medical University. 3. RESULTS 3.1. Three-dimensional structures of sea urchin spines. Sea urchin spines (Heterocentrotus mammillatus) (Figure 1a) have cylindrical shapes with a thickness of ~1 cm and lengths of several centimeters, which are primarily used for: locomotion through a ball-and-socket joint mode, sensing, as well as for defense against predators.28 The section view (Figure 1a) shows an original sea urchin spine with the crystallographic c-axis along the spine’s length and growth rings distributing perpendicular to the c-axis. SEM images of inner meso-structures (Figure 1b-d) show that the growth rings have relatively dense structures and open-cell stereoms consist of highly porous and interconnected structures. The growth rings ~80-100 µm thick mainly comprise a number of wedges that are connected through bridges (Figure 1b,c). The pore diameter of the open-cell stereoms is ~15-50 µm, and the strut thickness is ~6-25 µm (Figure 1d).

Fracture of the biogenic calcite from sea urchin spines was

shown in a conchoidal manner (Figure 1e), rather than a cleavage-like fracture of geological calcite. Moreover, the strut segment is similar to a configuration of a truncated conical shell (Figure 1f), which has zero mean curvature at every point on the surface, and therefore, can resist against an external load mainly through co-planar stresses.29 Thus, sea urchin spines with superior mechanical property are suitable for cutting and drilling to a variety of shapes, for example cylindrical samples with uniform holes as different bone implants (Figure 1g,h) in this study.


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Figure 1. The section view (a) of original sea urchin spines (Heterocentrotus mammillatus), SEM micrographs of inner structures of sea urchin spines (b-e), and configuration of a truncated conical shell for a strut in the sea urchin spine (f) and machined sea urchin spine samples as bone implants (g,h). 3.2. The relationship between mechanical property and mesostructures. Compressive strength for sea urchin spines can vary significantly among different specimens, due to non-uniformity in structures, such as difference in the number of



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growth rings and their distribution in samples, as well as a considerable number of other irregularities such as pre-existing flaws and microcracks (even macrocracks) in the structure.16 Weibull distribution of fracture strength (Figure 2) of original sea urchin spines and β-TCMP scaffolds after hydrothermal conversion at 200°C for two days were performed using the following equation (2):10 ఙ ௠

PሺVሻ = exp ቂ− ቀఙ ቁ ቃ బ

(2)

P(V) is the survival probability, σ0 and m are Weibull parameters obtained experimentally, and σ is the fracture strength. The Weibull parameters ‘m’ for the original sea urchin spines, and β-TCMP scaffolds after hydrothermal conversion at 200°C for 2 days are 3.10 and 1.92, respectively. The plots in Figure 2 indicate that the fracture strength of the original sea urchin spines at 50% fracture probability (P(V) = 0.5) is ~43.4 MPa, and for the resultant β-TCMP scaffolds is ~9.3 MPa; the latter being comparable to the fracture strength of human cancellous bone ~2-12 MPa.30,31 A previous study32 reported that the bend strengths of spines of sea urchin (Heterocentrotus trigonarius) varied between 13 and 41 MPa, and the average strength (σ50) was 26 MPa based on the Weibulll analysis. Moureaux et al.28 measured stiffness, hardness, Young’s modulus and bending strength for spines of sea urchin (Paracentrotus lividus) by nanoindentation, microindentation and bending tests, and showed that Young’s modulus and bending strength were 21.74 ± 3.59 GPa and 108.15 ± 24.82 MPa, respectively, on the skeleton shaft material. The above results again confirm that the mechanical strength of sea urchin spines vary from species to species, as well as from specimen to specimen for each species, consistent with our findings.


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Figure 2. Weibull distribution of fracture strength of (a) sea urchin spines (n = 33) and (b) resultant β-TCMP scaffolds (n = 27) after a hydrothermal reaction at 200°C for 2 days. FEA was performed to analyze the stress distribution in sea urchin spine samples under the compression loading (Figure 3). Micro-CT images illustrate the integral 3D interconnected structures of growth rings and the stereoms (Figure 3a). The average porosity of sea urchin spine specimens calculated using the buoyancy method was 60%-70%, comparable to that of human trabecular bone (porosity ~50%-90%).33 A sample section of 340 µm × 340 µm × 170 µm was constructed using finite element meshes consisted of 1,232,105 tetrahedrons (Figure 3b), and the distribution of von Mises stresses (Figure 3c), and a cross sectional view (Figure 3d) are presented. The red and blue regions represent the maximum and minimum values of stress, respectively. The results show that the major stress is concentrated along the dense growth rings, and dissipation of stress from the growth ring to struts in open-cell stereoms is clearly observed (Figure 3c,d), resulting in efficient energy absorption and high strength with lightweight structures.



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Figure 3. A micro-CT image of a sea urchin spine (a) and a selective meshing model for finite element analysis (b), and von Mises stress (c and d) distribution in the sea urchin spine sample under the compression loading with 0.5% strain (MPa). 3.3. Hydrothermal conversion of sea urchin spines to β-TCMP scaffolds. The powder XRD pattern (Figure 4a) confirms that the original sea urchin spine consists of Mg-calcite,22,23 and the diffraction peaks shift to larger angles in the pattern compared with a calcite phase without Mg doping, caused by substitution of Ca2+ ions by Mg2+ ions in the lattice. Sea urchin spines (calcium carbonate) can be converted into β-TCMP after hydrothermal conversion (Figure 4b). FTIR results of the original sea urchin spine and β-TCMP scaffold are shown in Figure 4c and Figure 4d, respectively. The spectrum from the original urchin spine shows characteristic bands at 710 cm-1 (carbonate γ4), 872 cm-1 (carbonate γ2) and 1408 cm-1 (carbonate γ3) resulting from calcium carbonate. Peaks from phosphate γ4 (604 cm-1) and phosphate γ3 (1022cm-1) are present in the spectrum of β-TCMP scaffold after a hydrothermal


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reaction at 200°C for 2 days, indicating that the conversion of calcium carbonate to calcium phosphate, consistent with previous results.16, 34

Figure 4. (a) Powder XRD pattern and (c) FT-IR spectrum of an original sea urchin spine, and (b) powder XRD pattern and (d) FT-IR spectrum of the sea urchin spine after hydrothermal conversion at 200°C for 2 days. Red lines in (a) and (b) indicate the diffraction peak positions for calcite (JCPDF# 05-0586) and β-TCP (JCPDF# 09-0169), respectively. SEM images show that bone-mineral-like nano-apatite particles formed on the surfaces of β-TCMP scaffold after soaking in simulated body fluid (SBF) for two weeks (Figure 5a), and assembled into a denser coating on the scaffold struts in three weeks (Figure 5b). Moreover, there was an approximately linear increase of sample weight by increasing the soaking time from one week to three weeks (Figure 5c), indicating progressive deposition of the apatite minerals. Apatite crystal formation and assembly are critical for bone regeneration in vivo.35 The formation of



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bone-mineral-like apatite layer suggests good bone integration ability of these β-TCMP scaffolds.27

Figure 5.

Apatite assembly on β-TCMP scaffolds soaked in SBF for (a) two weeks

and (b) three weeks, (c) the EDS result from the surface of a β-TCMP scaffold soaked in SBF for two weeks, and (d) the relationship between the increase of sample weight and soaking time. 3.4. Integration of newly-formed bone to open-cell structures. Micro-CT images showed that there was no evidence of spontaneous fusion of the rabbit femoral defect in the control group after one month (Figure 6a,b), whereas a small amount of newly-formed bone was found in the control group after three months, but only filling a small volume of the defect (Figure 6c,d). On the other hand, new bone formed on the surfaces of β-TCMP implants after one month (Figure 6e,f), while a larger volume of new bone was found after three months (Figure 6g,h).


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Figure 6. Micro-CT images of the control group (a-d) and the β-TCMP implant group (e-h) after implantation for one month (a,b,e,f) and three months (c,d,g,h). Newly formed bone (white color portions in f, h) were found on the surfaces of β-TCMP implants.



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Van-Gieson staining of the histological sections showed that the control group remained empty with only a small amount of newly-formed bone found post-operation for three months (Figure 7a-c), consistent with the previous micro-CT results. For the β-TCMP scaffolds (Figure 7d-i), direct bony apposition on the implants was found. There was no evidence of fibrosis tissue around the implants. These findings indicated that the β-TCMP implants showed great biocompatibility and underwent osseointegration. There was an increasing volume of regenerated bone growing into the open-cell β-TCMP structures with increase of implantation time from one to three months (Figure 7d-f). The electron dispersive x-ray (EDX) mapping of Mg element (Figure 7i) indicated the portion of the β-TCMP implant, considering that there was a significant amount of Mg in β-TCMP (also see Supporting Information Figure. S1d). EDX mapping of Ca element (Figure 7g) and P element (Figure 7h) show both the β-TCMP implant and regenerative new bone (also see Supporting Information Figure S1c). Thus, the majority of inner space of the β-TCMP implant was occupied by newly-formed bone in three months by comparing Figure 7i to Figure 7g,h. There was tight integration between the porous structures and newly formed bone (Figure 7f-h), which can improve the stability of the implants in vivo36 and reduce the risk of scaffold corruption and ceramic debris migration to adjacent soft tissues.


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Figure 7.

Van-Gieson staining for histological sections from the control group

(a-c), and the β-TCMP implant group (d-f) after implantation in rabbit femoral defects for one month (a, d), two months (b, e) and (c, f) three months, and EDS mapping of the implant in rabbit femoral defects for three months: (g) calcium (Ca) element distribution, (h) phosphorus (P) element distribution, (i) magnesium (Mg) element distribution. Mg spectrum (i) only existing in the β-TCMP struts shows the portion of the β-TCMP scaffold, while Ca (g) and P (h) spectra indicate both regenerative bone and β-TCMP scaffold portions. 3.5. In vivo degradation of open-cell β-TCMP scaffolds. The scaffold was implanted with a fusion Ti-6Al-4V device for fixation of beagle lumbar facet joints. The growth of newly-formed bone into the scaffold and the scaffold degradation were characterized by Micro-CT (Figure 8). The lumbar facet joint fusion was completed by either autogeneous bone (Figure 8a) or β-TCMP scaffold (Figure 8b) after seven months post-operation. There was an obvious degradation of the β-TCMP scaffold (Figure 8c) with the remnant volume/original scaffold volume ~22.8 ± 13.0%,



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whereas the volume percentage for the newly formed bone/total ROI was about 51.0 ±4.6%. The β-TCMP scaffold was nearly completely degraded and replaced by newly-formed bone after implantation for ten months (Figure 8e,f), which also achieved complete fusion similar to that by autogeneous bone (Figure 8d). The volume percentage for the newly formed bone/total ROI was approximately 54.8±3.5% after ten months implantation.

Figure 8.

Micro-CT images of Ti6Al4V cages with autogenous bone (a, d) and

β-TCMP scaffold (b, c, e, f) for fixation of beagle lumbar facet joints at seven months (a-c) and ten months (d-f). The bony tissues were removed from the images (c, f) by setting a threshold of contrast filtration. SEM images of implants (Figure 9) indicated that newly-formed bone grew along the β-TCMP scaffold and completed the fusion at seven months (Figure 9a-c, and also see Supporting Information Figure S2). The partial enlargement showed the growth of newly formed bone into the pores of the implant, and there was tight integration between regenerative bone and the β-TCMP scaffold (Figure 9d). Obvious disruption of the porous structures suggesting early degradation was also observed. There were only a small amount of β-TCMP residue in the middle of the fusion cage after implantation for ten months (Figure 9e,f), suggesting a progressive degradation


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of the β-TCMP scaffold from the outside to inside. The partial enlargement images (Figure 9g,h) showed obvious replacement of the β-TCMP struts by newly-formed bone. These results indicated that there was a good match between β-TCMP scaffold degradation and new bone formation.

Figure 9.

SEM images of Ti-6Al-4V fusion cages with β-TCMP scaffolds after

implantation for seven months (a-d) and ten months (e-h). (a, e) the section view of



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Ti-6Al-4V fusion devices, (b, f) growth of newly formed bone into the porous structures of β-TCMP scaffolds, (c, g) integration between newly formed bone and β-TCMP scaffolds, d) and h) replacement of β-TCMP scaffolds by regenerative bone. Red dash circles (a, b, e) show integration of the β-TCMP scaffolds and newly formed bone, whereas yellow dash lines (c, g) indicate the tight boundary. The yellow dash circle (d) shows the infiltration of new bone into the porous scaffold, and the orange dash circles (g, h) indicate the remaining β-TCMP struts.

4. DISCUSSION Calcium phosphate bioceramics have been widely used as scaffolds for bone defect repair due to their good biocompatibility and osteointegration property.13 The porosity, pore size and interconnectivity play important roles in osteoconductivity and biodegradability of these bioceramics.37 For example, biogenic calcium carbonate (CaCO3) skeletons from corals18 and cuttlebones38 have previously been used to produce porous HAP bioceramics through hydrothermal reactions for bone defect repair considering that these natural skeletons show high porosity, large pore size and great interconnectivity, suitable for ingrowth of new bone and blood vessels. However, these bioceramics have relatively poor mechanical strength at high porosity,39 potentially leading to corruption during implantation and migration of the resulting ceramic debris to adjacent soft tissues.40 Machinability, to shape these brittle porous scaffolds is extremely difficult, restricting their use as small bone defect repair. Moreover, HAP bioceramics converted from corals and cuttlebones are not biodegradable, which can remain in sites for years after implantation.41 In this study, sea urchin spines (Heterocentrotus mammillatus), with hierarchical open-cell structures, similar to human trabecular bone, with superior mechanical strength suitable for machining to shape, are explored for potential applications to bone defect repair. In nature, cellular structures are most commonly surrounded by dense walls, such as bones, sea urchin spines, and quills, forming sandwich or layered


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structures, which show superior mechanical properties with high strength-to-weight ratios.42 For example, the layered mesostructures combining thin solid shells with core lightweight foams from sea urchin spines can provide effective resistance to local buckling (crimping), similar to animal antlers and skeletal bones43. A previous study44 showed that the crushing strength to weight ratio of spines from three species of sea urchin, Stylocidaris affinis, Heterocentrotus mammillatus and Eehinometra mathaei, was equivalent to or greater than that of mollusc skeletons and most calcareous rocks. The simulation analyses (Figure 3) also confirmed that the hierarchical mesostructures of sea urchin spines were key to dissipation of stress from the growth ring to struts in open-cell stereoms, resulting in efficient energy absorption and high strength with lightweight structures. In addition, fracture of the biogenic calcite from sea urchin spines was shown in a conchoidal manner (Figure 1e), rather than a cleavage-like fracture of geological calcite. Seto et al.20 suggested that the residual surface layer of ACC and/or macromolecules remained around the nanoparticle units of the mesocrystal structure and contributed to the conchoidal fracture behavior. Thus, sea urchin spines showing superior mechanical properties are suitable for machining to shape on demand for a variety of bone graft applications, including bone defect repair, spinal fusion and maxillofacial reconstruction. Besides the advantage of superior mechanical property, sea urchin spines can be hydrothermally converted to biodegradable β-TCMP, despite many other calcium carbonate skeletons from corals, cuttlebones, and seashells were converted to non-degradable HAP after hydrothermal reactions.18 The high content of Mg2+ ions in the calcite mineral from sea urchin spines, but not in the aragonite mineral from corals, cuttlebones or seashells, likely results in different final products via different hydrothermal reactions. Sea urchin spines mainly comprise of magnesian calcite crystals that generate via the initial deposition of amorphous calcium carbonate with high amounts of Mg2+ ions.23 The concentration of Mg element in calcite is influenced not only by environmental factors, such as water temperature, but also by physiological factors.45-48 In addition, organic macromolecules play important roles in



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the calcite crystal growth as well as the formation of mesostructures of sea urchin spines.20,21,23 There was a high amount of Mg for substitution of Ca in the calcite crystals (Mg/(Mg+Ca) = 11.53±0.68 mol.%) from original sea urchin spines based on the inductively coupled plasma mass spectrometry (ICP-MS) tests. The formation of β-TCMP, not hydroxyapatite, by the hydrothermal reaction was likely due to the relatively lower solubility of β-TCMP with a great amount of Mg (Mg/(Mg+Ca) > 10 mol.%) than hydroxyapatite (HAP) as well as the inhibition effect of Mg2+ ions on HAP crystal formation.49,50 Porous structures, especially pore size and connectivity, of the scaffolds, play important roles in cell colonization and bone ingrowth (osteoconductivity). Scaffolds for bone defect repair should have interconnected pores with a high porosity to support cell penetration, vascular ingrowth and nutrient transportation as well as waste product elimination. In addition, macro-pores (> 100 µm) show substantial bone ingrowth, meso-pores (50–100 µm) result in ingrowth of un-mineralized osteoid tissue, and micro-pores (10–50 µm) were infiltrated only by fibrous tissue.14,51,52 However, most porous scaffolds used in the previous studies showed closed-cell structures and pores were not well interconnected, which may prevent further growth of bony tissue into the inner spaces. It was found here that β-TCMP scaffolds maintained the mesostructures of original sea urchin spines after hydrothermal conversion. Newly-formed bone grew into the porous structures of β-TCMP scaffolds with an average pore size ~15-50 µm, and occupied the majority of inner space after implantation in rabbit femoral defects for three months (Figure 7f-h). These results suggest that open cellular structures and significant interconnectivity from sea urchin spines53,54 are beneficial to bone ingrowth, compared to the bioceramics with poor pore connectivity. Degradation of bioceramics are crucially important to bone defect repair. Slow degradation (e.g. HAP ceramics) prevents formation of new bone,55,56 while fast degradation (e.g. calcium carbonate and calcium sulfate) leads to formation of fibrous


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tissue and prevents further densification of new bone tissue.57,58 The β-TCP bioceramics showed suitable degradability rates compared with HAP and calcium carbonate ceramics.59 However, degradation of small macroporous β-TCP granules was generally found to be much faster than the formation of new bone when used for bone defect repair,60,61 likely due to corruption of β-TCP granules during degradation. The β-TCMP scaffolds produced from sea urchin spines that can be machined into particular shapes at certain sizes may provide advanced solutions to such limitation of small β-TCP granules. Indeed, degradation of β-TCMP scaffolds matched well with new bone formation during seven-month implantation for fixation of beagle lumbar facet joints (Figure 8b,c and Figure 9a-d), which persisted till ten months (Figure 9e-h). The good degradability of the β-TCMP scaffold is largely because of the unique structures of sea urchin spines, which have open-cell structures with thin struts (~6-25 µm thick). The open-cell structures with proper pore size (~15-50 µm) allow infiltration of cells and nutrients to facilitate new bone formation, and the thin struts can be easily degraded by osteoclasts to provide more spaces for further tissue ingrowth. Therefore, the unique structures of sea urchin spines with open-cell structures and thin struts are key to facilitating scaffold degradation and new bone formation. These findings may provide new insights to design of bio-inspired functional materials particularly with open-cell structures and thin struts, possibly fabricated using advanced 3D printing technique,62 to obtain superior mechanical strength as well as great match between scaffold degradation and new bone formation. 5. CONCLUSIONS Sea urchin spines comprising hierarchical mesostructures of open-cell stereoms surrounded by dense growth rings have superior mechanical strength, which can be machined into particular shapes. The FEA results revealed that the growth rings mainly withstood the large compression load, while the open-cell stereoms can dissipate the loading energy, leading to high strength-to-weight ratios. The β-TCMP scaffolds by hydrothermal conversion of sea urchin spines retained the original



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mesostructures and showed mechanical strength comparable to human trabecular bone. New bone grew into the open-cell structures of β-TCMP scaffolds, which were mostly filled after implantation in rabbit femoral defects for three months forming tight interface between the newly formed bone and the scaffolds. The thin struts can be easily degraded in vivo, which further facilitate the ingrowth of new tissue. The β-TCMP scaffolds were nearly completely replaced by newly formed bone in ten months, when used for fixation of beagle lumbar facet joints together with Ti-6Al-4V fusion cages. These results suggest that β-TCMP scaffolds, converted from sea urchin spines, while retaining the hierarchical mesostructures, are ideal scaffolds for bone regeneration, which show advantages of good mechanical strength and suitable biodegradability. These findings can inspire the design of new lightweight open-cell materials for bone defect repair or replacement. ASSOCIATED CONTENT Supporting Information Including Figure S1 for SEM images of implants in rabbit femoral defects for 2 months and EDS results to show the regenerative bone, and Figure S2 for an SEM image and EDS results of a β-TCMP scaffold fitted in a Ti-6Al-4V fusion cage after implantation for 7 months. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Xing Zhang), [email protected] (Zheng Guo), [email protected] (Yue Zhu). Author Contributions #

These authors contributed equally to this work.


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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (No. 31300788, No. 81171773), the National High-tech R&D Program of China (863 program, No. 2015AA033702), the Hundred-Talent Program from Chinese Academy of Sciences. We acknowledge National Supercomputing Center in Shenzhen for providing the computational resources and ANSYS software. We also thank Dr. Shaogang Wang, Dr. Xingxing Zhang and Yankun Zhu at Shenyang National Laboratory for Materials Science for help with micro-CT analysis and discussion of finite element analysis. REFERENCES (1) Gibson, L. J.; Ashby, M. F. Cellular Solids: Structure and Properties, 2nd ed.; Cambridge University Press: UK, 1997. (2) Schaedler, T. A.; Jacobsen, A. J.; Torrents, A.; Sorensen, A. E.; Lian, J.; Greer, J. R.; Valdevit, L.; Carter, W. B. Ultralight Metallic Microlattices. Science 2011, 334 (6058), 962-965. (3) Zheng, X.; Lee, H.; Weisgraber, T. H.; Shusteff, M.; Deotte, J.; Duoss, E. B.; Kuntz, J. D.; Biener, M. M.; Ge, Q.; Jackson, J. A.; Kucheyev, S. O.; Fang, N. X.; Spadaccini, C. M. Ultralight, Ultrastiff Mechanical Metamaterials. Science 2014, 344 (6190), 1373-1380. (4) Duan, G.; Jiang, S.; Jerome, V.; Wendorff, J. H.; Fathi, A.; Uhm, J.; Altstaedt, V.; Herling, M.; Breu, J.; Freitag, R.; Agarwal, S.; Greiner, A. Ultralight, Soft Polymer Sponges by Self-Assembly of Short Electrospun Fibers in Colloidal Dispersions. Adv. Funct. Mater. 2015, 25 (19), 2850-2856. (5) Maiti, A.; Small, W.; Lewicki, J. P.; Weisgraber, T. H.; Duoss, E. B.; Chinn, S. C.; Pearson, M. A.; Spadaccini, C. M.; Maxwell, R. S.; Wilson, T. S. 3D Printed Cellular



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Lightweight Open-Cell Scaffolds from Sea Urchin Spines with Superior

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