âSteelâConcreteâ Inspired Biofunctional Layered Hybrid Cage for

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A “steel-concrete” inspired biofunctional layered hybrid cage for spine fusion and segmental bone reconstruction Jingzhou Yang, Yu Shrike Zhang, Pengfei Lei, Xiaozhi Hu, Mian Wang, Haitao Liu, Xiulin Shen, Kun Li, Zhaohui Huang, Juntong Huang, Jie Ju, Yihe Hu, and Ali Khademhosseini ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00666 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 7, 2017

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ACS Biomaterials Science & Engineering

A “steel-concrete” inspired biofunctional layered hybrid cage for spine fusion and segmental bone reconstruction Jingzhou Yang†,‡,§, Yu Shrike Zhang‡,§,∑*, Pengfei Lei‡,∏,#, Xiaozhi Hu†*, Mian Wang‡,§,∆, Haitao Liu¶, Xiulin Shen¶, Kun Li∏, Zhaohui Huang¶, Juntong Huang∞, Jie Ju‡,§, Yihe Hu∏*, and Ali Khademhosseini‡,§,∑,Ø* †

School of Mechanical and Chemical Engineering, University of Western Australia, 35 Stirling Highway, Perth, Western Australia 6009, Australia ‡ Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, United States § Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ∑ Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, Massachusetts 02115, United States ∏ Orthopedics Department, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, People’s Republic of China # Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, United States ∆ School of Chemistry and Chemical Engineering, Guangxi University, 100 University East Road, Nanning, Guangxi 530004, People’s Republic of China ¶ School of Materials Sciences and Technology, China University of Geosciences, 29 Xueyuan Road, Beijing 100086, People’s Republic of China ∞ School of Materials Science and Engineering, Nanchang Hangkong University, 696 Fenghe Nan Street, Nanchang, Jiangxi 330063, People’s Republic of China Ø Department of Physics, King Abdulaziz University, Abdullah Sulayman Street, Jeddah 21569, Saudi Arabia

ABSTRACT In this paper we report a “steel-concrete” inspired layered hybrid spine cage combining titanium mesh and bioceramic scaffold, which were welded together through a bioglass bonding layer using a novel multi-step manufacturing methodology including three-dimensional slip deposition, gel casting, freeze drying, and co-sintering. The interfacial welding strength achieved 27 ± 0.7 MPa, indicating an excellent structural integrity of the hybrid cage construct. The biocramic scaffold layer consisting of wollastonite and hydroxyapatite had an interconnected,

ACS Paragon Plus Environment

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highly porous structure with a pore size of 100-500 µm and a porosity of >85%, well fufilling the structural requirements of bone regeneration. Simulated body fluid immersion assay showed that the hybrid cage exhibited excellent biodegradability to facilitate rapid bone-like apatite formation. In vitro studies demonstrated that the hybrid cage supported attachment, spreading, growth, and migration of bone/vessel-forming cells and triggered osteogenic differentiation of human mesenchymal stem cells. In vivo studies further suggested that the titanium-bioceramic hybrid cage could actively promote fast generation of new bone tissues within 12 weeks of implantation in a rabbit femoral condyle model. This study has provided a new design and fabrication methodology of hybrid cages by integrating strong mechanical properties with excellent biological activities including osteoinductivity and bone regeneration ability, for spine fusion and segmental bone reconstruction.

KEYWORDS:

Hybrid spine cage; titanium-bioceramic bone scaffold; wollastonite;

hydroxyapatite; osteoinductivity; mesenchymal stem cells

1. INTRODUCTION Spine vertebrae and intervertebral disk defects, which often result from trauma (e.g. battles and accidents) or diseases (e.g. osteoarthritis, spondylosis, osteonecrosis, tumor, degeneration, and infection), often lead to spine dysfunction and complications including nerve root or spinal cord compression, progressive neurological deficits, intractable pain, and sometimes even permanent disability or death 1-3. Spinal arthrodesis (spine fusion) is deemed a necessary surgery to fuse two or more vertebrae for decompressing the affected nerve roots and spinal cord, and restoring biofunctions of the spine 1, 4. There are over 300,000 spine fusion surgeries performed in the United States every year

5-6

. The first-choice material for spinal fusion is autologous bone

graft that exhibits excellent mechanical properties and ideal biological properties including


2

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osteoconductivity and osteoinductivity, but severe drawbacks such as donor-site morbidity, graft collapse, and supply limitation cannot be disregarded 1-2. As an alternative, titanium cages have been used for decades for spine fusion because of their good mechanical properties (e.g. immediate stability in compression, resisting high-cycle displacement, maintaining spinal alignment and foraminal intervertebral height), high corrosion resistance, and biocompatibility 3, 7-11. However, titanium as a bioinert material, is not optimal for this application, because it is not osteoconductive nor osteoinductive to form strong integration with host bone or other tissues surrounding the cage

12

. Therefore spine fusion failures with

titanium cage implantation still occur, especially in the case of multi-level corpectomy where the failure rate can be as high as over 50% 13-15. In addition for use as spine fusion implants, titanium cages have been adopted for repair of load-bearing segmental long bone (e.g. femur, tibia) defects 16-21

, but the lack of sufficient bioactive properties makes solid bony fusion and satisfactory bone

regeneration difficult to achieve. To improve the biological properties of titanium cages, orthopedic surgeons have filled titanium cages with autologous bone segments or bioceramic granules

8, 11, 22-24

. Nevertheless, the bone segments and bioceramic granules are packed loosely

and have limited bonding with the titanium cages so that the structural integrity is usually insufficient. On the other hand, while dense bioceramic/glass coating could improve the bioactivity of titanium implants 25-26, these coatings are oftentimes not amenable to cell migration, nutrient transport, and bone/vessel ingrowth that are critical for bone fusion and regeneration. In this paper we propose a new design of bioactive hybrid spine cage system inspired by the “steel-concrete” structure, consisting of a titanium mesh and a highly macroporous bioceramic scaffold to integrate both outstanding mechanical properties and strong biological activities. Hydroxyapatite (HA) has been well documented to possess excellent bioactivity, biocompatibility, osteoconductivity, and in vivo bone-forming ability

27-31

. Wollastonite (WS), a typical silicate

mineral, has attracted increasing attentions in recent years due to its outstanding osteoinductivity,


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fast biodegradability, biomineralization ability, and angioinductivity

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32-36

. The use of natural WS

(nWS) for bone repair applications has also been studied, suggesting that nWS not only is biocompatible but also has better biodegradability and bone-forming ability than synthetic WS 3739

. Therefore, here we combined HA and nWS in the composite bioceramic scaffold to achieve

optimal bioactive properties. A unique multi-step manufacturing methodology combining threedimensional (3D) slip deposition, gel casting, freeze drying, and co-sintering, was developed to fabricate an innovative functional hybrid cage. The hybrid structure was characterized for its porosity, pore size, and interfacial bonding state. In vitro studies were subsequently conducted to evaluate the biological properties including bone/vessel cell-hosting ability, bone-like apatite formation, and osteoinductivity. In vivo evaluation was finally carried out to investigate the osteoconductivity and bone regeneration capability.

2. MATERIALS ANF METHODS 2.1 Materials The key materials are as follows: 45S5 Bioglass (500 nm, Guijian Biomedical Materials Co., Shanghai, China), HA (200 nm, Sigma-Aldrich Ltd., MO, USA), nWS (5 µm, Jiangxi Huan Yu Wollastonite Mineral Materials Co., Ltd., Jiangxi, China), and commercial pure titanium mesh with a herringbone pattern (1 mm in both grid thickness and space between two grids) (Baoji INT Medical Titanium Co., Ltd., Shanxi, China). Additional raw materials and chemicals include Dolapix (Zschimmer & Schwarz-Gmbh, Lahnstein, Germany), gelatin (Ward Mckenzie Ltd., Victoria, Australia), ethanol, and distilled water. Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS), α-minimum essential medium (α-MEM), fetal bovine

serum

LIVE/DEAD®

(FBS),

penicillin-streptomycin

(Pen-Strep)

antibiotic

solution,

trypsin,

viability/cytotoxicity kit (calcein-AM, green and fluorescent ethidium

homodimer-1, red), and phalloidin, 4’,6-diamidino-2-phenylindole (DAPI), human mesenchymal


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stem cells (MSCs), MC3T3-E1 preosteoblasts, human umbilical vein endothelial cells (HUVECs), and alkaline phosphatase (ALP) staining kit were purchased from ThermoFisher (NY, USA). Endothelial cell basal medium (EBM) was purchased from Lonza (MA, USA). All other chemicals were purchased from Sigma-Aldrich (MO, USA) unless otherwise noted.

2.2 Fabrication of “steel-concrete” structured titanium-bioceramic hybrid cage The large difference in the physical and chemical properties between the titanium mesh and the bioceramic scaffold makes it very challenging to combine them together as an integral hybrid cage construct. To this end, we have developed a unique processing method by combining 3D slip deposition, gel casting, freeze drying, and co-sintering. Titanium has a coefficient of thermal expansion (CTE) of 8.6 × 10-6/K, and HA has a much higher CTE of 15.2 × 10−6/K therefore used nWS microfibers (8.1-9.2 × 10−6/K

40

40

. We

) to reduce the mismatch of CTEs between

the titanium mesh and bioceramic scaffold. We use the equation αC= αM (αp/αM)PVF

41-42

to

calculate the CTE of the composite bioceramic layer. αC—CTE of the composite; αM—CTE of the matrix; αP—CTE of the second phase; PVF—ratio of the second phase added into the matrix. The equation is based on dense materials. Our previous study showed that a porous structure could reduce the CTE mismatch and shrinkage during co-sintering 42-45. Through calculation and experimental optimization, we found half of the value from the equation worked better. Finally we chose a composition of the bioceramic scaffold consisting of 40 wt% nWS and 60 wt% HA. It should be mentioned that, the nWS, natural wollastonite microfibers may be replaced with synthesized ones that have similar chemical and phase compositions. They both possess outstanding osteoinductivity, fast biodegradability, biomineralization ability, and angioinductivity 32-36

. The difference is that, the natural WS with more glassy phase, has better biodegradability

and bone-forming ability than synthetic WS

37-39

. Also the nWS is more cost effective. To

achieve a strong interfacial bonding between the titanium mesh and bioceramic scaffold, a


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bioglass welding layer was further coated onto the surface of the titanium mesh to interface with the bioceramic microstructure. The low-density slip was prepared using ethanol as the solvent. The solid content of bioglass was 10 wt%, where 20 wt% poly(methyl methacrylate) (PMMA) microparticles (20-50 µm) were added as pore generators and 0.5 wt% Dolapix was employed as the dispersant. The bioceramic gel was made with gelatin and distilled water heated at 60-80 °C for 3-5 mins, where the solid content of mixed nWS-HA bioceramic particles was 25 wt%. Titanium mesh cage was first completely cleaned by ultrasonication in ethanol and then dipped in the bioglass slip for 1-3 s. The bioglass coating was allowed to dry at room temperature. Such dipping and drying processes were repeated for 20-30 times to achieve a required thickness for the bonding layer. The titanium mesh coated with the bioglass bonding layer was then placed in a plastic mold and casted with the bioceramic gel. After 30 min, the sample was retrieved and frozen at -20 °C for 2 days. Freeze drying was subsequently carried out at -40 °C in the vacuum of 100 µm could form in the bioceramic scaffold. No noticeable shrinkage took place during freeze-drying, and we could therefore obtain a crack-free hybrid cage in its original shape. The final co-sintering was carried out at 900 °C for 10 min in a furnace with vacuum atmosphere to generate the titanium meshbioceramic hybrid cage. This unique procedure is material-independent and can be expanded to any other metal-ceramic systems to obtain “steel-concrete” structured constructs with millimeterthick highly porous ceramic coatings.

2.3 Mechanical testing To evaluate the structural integrity of the hybrid cage, the bioglass welding strength between titanium mesh and nWS-HA bioceramic scaffold was determined with the method shown in ASTM International C633-01 using an Instron 5982 machine. The 15 × 5 (D × H) mm2


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cylindrical titanium samples with bioglass welding layer coatings (100-200 µm) were employed for layer-layer interfacial bonding strength determination. Two steel rods with a diameter of 10 mm were used to connect the sample and the fixture. The bonding agent was Araldite super strength glue. The loading strain rate was 0.1 mm/min. The welding strength was calculated by maximum load/contact area.

2.4 Microstructure, phase composition, and porosity characterization The microstructural and composition details, such as the titanium-bioceramic interface and macroporous structure of the nWS-HA bioceramic scaffold were observed by a Zeiss 1555 scanning electron microscope (SEM, Carl Zeiss Co., Oberkochen, Germany) with an accessory energy dispersive spectroscopy (EDS). The distributions of major elements of the hybrid cage were analysed across the titanium mesh, reaction layer, bioglass welding layer, and the bioceramic scaffold, by element mapping and line scanning. In order to investigate the interfacial structure of the hybrid cage, the samples were fixed within epoxy resin and well grounded and polished with diamond suspensions to obtain a smooth surface. The resin mounting was essential to avoid damage of the macroporous structure during polishing. Phase compositions of the sintered bioceramic scaffold was examined with X-ray diffraction (XRD, Shimadzu Co., Kyoto, Japan) at a scanning speed of 1.2 °/min (CuKa1 radiation). The porosity was measured by Archimedes’ principle. The porosity calculation equation was p= ((m3-m1)/(m3-m2)) × 100%, where p = apparent porosity, m1 = dry mass measured in the air, m2 = wet mass measured in the liquid, and m3 = wet mass (after fully absorbing of immersion liquid) measured in the air. Statistical values were generated based on six samples. Lubricant oil vacuum immersion was carried out for 1 h to obtain samples for measuring m2 and m3.

2.5 Biomineralization assay


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The hybrid cage samples were soaked in 50 mL of simulated body fluid (SBF) to investigate their biomineralization ability in terms of bone-like apatite formation. The SBF was replaced daily to maintain constant ion concentrations. The experiment was carried out at 37 °C in an incubator. After immersion for 1 day, the samples were gently rinsed with distilled water for five times followed by air drying at room temperature. The phase composition of biomineralized apatite film on the hybrid cage surface was determined by XRD. The phosphorus, calcium and silicon concentrations in the SBF were examined directly after the samples were taken out on days 1, 3, 5, 7, 11, and 13 by using an inductively coupled plasma atomic emission spectrometer (ICP-AES, ICPS-8100, Kyoto, Japan). The morphology of biomineralized apatite was observed with an SEM.

2.6 In vitro cellular response with bone/vessel-forming cells To evaluate the cell-material interaction and in vitro osteogenesis/angiogenesis potential, three types of cells including MSCs, MC3T3 preosteoblasts, and HUVECs were used. To keep the constant osteogenic differentiation capability, we used passage 3 of MSCs for all the experiments. The sample size was 5 × 5 × 2 mm3. The culturing conditions were maintained at 5% CO2, 95% humidity, and 37 °C. α-MEM, DMEM, and EBM with essential antibiotics and supplements were used for culturing MSCs, MC3T3 preosteoblasts, and HUVECs, respectively. When the cells proliferated to 85-90% confluence in flasks, they were passaged. The samples were sterilized by soaking in 70 % ethanol for 2 h and exposure to UV light for 1 h, followed by PBS washing for 5 times and immersion in medium for overnight. Next, 50,000 cells in 100 µL medium were gently seeded onto each sample in a 24-well plate and incubated at 37 °C for 2-3 h to allow cells to attach. New medium was then added for subsequent culture. The medium was changed every 2 days. On days 1, 4, and 7, live and dead staining was carried out. On day 7 and day 14, f-actin/nuclei staining was conducted. The samples were observed under a fluorescence


8

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microscope and a confocal laser scanning microscope (CLSM, Carl Zeiss Co.) to study the cell viability, attachment, morphology, spreading, migration, and cytoskeletal organization. To visualize the cell adhesion and biomineralized calcium aggregates, SEM was used to observe the sample surface with MSCs cultured for 28 days. The samples were placed in a 12-well plate. Then the samples were dehydrated using graded ethanol concentrations of 50%, 70%, 80%, 90%, and 100% (v/v), and further dried in air overnight. The samples were sputter coated with gold prior to SEM observation.

2.7 Osteoinductivity evaluation To investigate the osteoinductivity of the titanium-bioceramic hybrid cage, ion extract dilutions were prepared for culturing MSCs. HA ceramic was used as the control material. 1 g of fine nWS-HA ceramic powder was soaked in 5 mL of α-MEM and incubated at 37 °C for 2 days; the mixture was centrifuged to collect the supernatant; after sterilization with a filter (Millipore, 0.22 µm), the original extracts were diluted with complete medium by 64 times, and stored at 4 °C for further use. MSCs were then cultured in the extract dilutions for 28 days to study the osteogenic differentiation. Phalloidin/DAPI staining was conducted at day 7 to visualize the cytoskeleton and nuclei and to study the cell spreading and distribution. ALP staining was carried out to determine the early osteogenic differentiation of MSCs. Semi-quantitative study was conducted to obtain the percentage of differentiated cells. Six images were randomly taken and the percentages of purple areas (ALP-positive cells) were calculated with ImageJ. The ALPpositive cells were in purple color so that they could be distinguished from ALP-negative cells. We also counter-stained the nuclei of the cells to obtain their total numbers. Alizarin Red S (pH=4.2) staining was conducted to study the mineralization by mature osteoblasts. The ALPand Alizarin Red-stained samples were observed macroscopically (digital camera) and microscopically (ECLIPSE Ti-S optical Inverted microscope, Nikon, Japan). To quantify the


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mineralization, the Alizarin Red stain was extracted by dissolving in 1.5 M acetic acid solution for overnight. Then the extract was transferred to an Eppendorf tube and vortexed for 2 min. After that the extract was heated at 80 °C for 10 min followed by ice cooling for 5 min. The Eppendorf tube was centrifuged for 15 min at 20000 X g. Afterwards 400 µL of supernatant was mixed with 550 µL 0.2 M NaOH solution to adjust the pH value to 4.2. Then 100 µL of the solution was transferred to each well of a 96-well plate. The absorbance measured at 405 nm in a plate reader (ThermoFisher) for quantitative determination of mineralization.

2.8 Preliminary in vivo osteoconductivity and bone formation assessment Adult New Zealand white rabbits (pathogen-free, mean weight, 2.2 ± 0.2 kg) were used for in vivo studies (femoral condyle defect model) with the approval of Animal Welfare Committee of Xiangya hospital, Central South University of China. After anesthesia by subcutaneous injection of ketamine (36 mg/kg) and xylazine (5mg/kg), 2-cm longitudinal skin incisions were generated at the surfaces of the femur condyles. A cylindrical defect was made on the side of the condyle using an orthopedic drill with a pit size of 5 mm in diameter. The bone defects were implanted with steam-sterilized small samples (D × H = 5 × 8 mm2). Then the muscle and skin were irrigated and closed in layers using synthetic absorbable sutures and antibiotic prophylaxis provided. After recovery, the rabbits were kept in individual cages on free-range pasture and fed with a normal diet. The regions of interest (ROIs) were harvested at 12 weeks post-implantation, after humane euthanasia. Reclaimed ROIs were carefully dissected. The ROIs were decalcified in ethylenediaminetetraacetic acid (EDTA) and then fixed in 4% formaldehyde. After that the ROIs were thoroughly rinsed in PBS, dehydrated in a graded series of ethanol solution from 50% to 100%, cleared in xylene, and mounted in paraffin wax. Sections with a thickness of 5 µm were obtained for hematoxylin-eosin (H&E) and osteocalcin staining according to the standard


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protocols. The stained sections were observed under an optical microscope and photos were taken for histological analysis.

2.9 Statistical analysis Statistical significance was determined for replicates of six by student t-test or one-way ANOVA (ANalysis Of VAriance) with post-hoc tukey HSD (Honestly Significant Difference) test. All data collected were presented as mean ± standard deviation (SD) of six samples. A Pvalue of 0.05 or less was deemed statistically significant.

3. RESULTS AND DISCUSSION The hybrid spine cage was composed of a titanium mesh and a macroporous bioceramic scaffold welded by a bioglass bonding layer, as shown in Figure 1. In our “steel concrete” inspired design concept, the titanium mesh provides mechanical functions to share biomechanical load and distribute stress, while the bioceramic scaffold gives biological functions to host bone/vessel-forming cells, deliver bioactive factors, preserve tissue volume, and promote osseointegration and new bone formation. Modern spine cages normally have a hollow structure in the center and a thin wall containing macroscale pores to provide areas for maximum bone ingrowth and facilitate radiographic assessment of bony intergration and bone regeneration

5, 46

. In

this study, we used a thin-wall titanium mesh tube to mimic the spine cage that provided the basic mechanical support for further functionalization, with an outer layer of tightly bond macroporous bioceramic scaffold to integrate biological functions and form a biofunctional hybrid cage system.


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Figure 1. The “steel-concrete” inspired titanium mesh and bioceramic scaffold hybrid cage integrates mechanical and biological properties for spine fusion. The strong and tough titanium mesh provides structural function to share biomechanical load and distribute stress. The highly macroporous composite biceramic scaffold anchored onto the titanium mesh offers biological function to host bone-forming cells, allow vasculature ingrowth, enable cell migration and nutrient transport, and relase osteoinductive inorganic ions to facilitate bone regeneration.


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Figure 2. Manufacturing of titanium-bioceramic hybrid spine cage using a novel multi-step method. (a) The processing steps include low-density slip deposition (Step 1), air drying (Step 2), gel casting (Step 3), pre-freezing (Step 4), freeze drying (Step 5), and vaccum co-sintering (Step 6). (b) Photographs showing the hybrid cage at various key processing steps: original titanium mesh cage, and that after slip deposition, gel casting, freeze-drying, and co-sintering (from left to right). (c-h) Microstrcture characterization of the hybrid cage: (c) Secondary electron (SE2) SEM image showing the appearance and mesh grid thickness (1 mm) of the original titanium mesh; (d) Back scattered diffraction (BSD)-SEM image showing titanium mesh with slip-deposited


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bioglass bonding layer; (e) BSD-SEM image showing the hybrid cage after freeze drying and cosintering; (f, g) enlarged BSD-SEM images showing the microstructre of the hybrid cage and the interface between bioceramic scaffold and titanium mesh; (h) EDX elemental mapping results showing the bonding layer containing titanium, silicone, and oxygen. Scale bars: b, 10 mm; c-f, 500 µm; g,h, 100 µm.

To manufacture such a titanium-bioceramic scaffold hybrid cage, we developed and optimized a unique multi-step processing methodology of 3D slip deposition freeze drying, and co-sintering

44

43, 47

, gel casting,

(Figure 2a). We combined HA and nWS in the bioceramic

scaffold to achieve excellent osteoconductivity, osteoinductivity, and in vivo bone-forming ability 27, 34-35, 48

. Generation of tough bonding between the the titanium mesh and the macroporous

bioceramic scaffold have been historically very challenging due to the distinctive physical and chemical properties between the two materials. Therefore, in the first step we deposited a bioglass-based bonding layer onto the surface of the titanium mesh by 3D low-density slip deposition. Slip deposition is a type of deposition that involves direct transfer of a slurry of particles onto the surface. The ethanol-based slip contained low concentrations of bioglass powder and polymer particles. It is critical to note that, to avoid cracking and delamination of the bonding layer caused by drying stress we repeated the dipping/drying procedure for an optimum of 20-30 cycles. Then the titanium mesh coated by bioglass was gel-casted in a mold. This waterbased gel contained bioceramic particles (a mixture of HA and nWS) and gelatin. The gel-casted titanium-bioceramic construct was pre-frozen to allow for the growth of ice crystals in the gelceramic. The next step relied on freeze drying to generate macropores and channels in the bioceramic scaffold. The last step featured co-sintering in vacumm to solidify the bonding layer and the bioceramic scaffold, as well as to obtain the hybrid cage with good structural integrity. It should be pointed out that our novel methodology based on gel casting and freeze drying can


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surpass the coating thickness limitation of the conventional coating-based techniques so that we were able to fabricate such “steel-concrete” structured titanium mesh and bioceramic scaffold hybrid cages (Figure 2b).

Figure 3. Microstructure features and phase composition of the bioceramic scaffold on the hybrid cage. The bioceramic scaffold had a macroporous structure with the pore size of a few hundred micrometers, where elongated nWS grains and equiaxed HA grains were interlocked together. SEM images showing (a,b) the interior structure, and (c,d) the surface structure. (e) XRD pattern confirming that the sintered bioceramic scaffold contained both nWS and HA. (f) EDS analysis showing the major elements in the nWS grains. Scale bars: a,c 200 µm; b,d 10 µm.

Figure 2c-g shows the microstructure of the developed hybrid spine cage. The thickness of the titanium mesh grid was 1 mm (Figure 2c). The microporous bioglass bonding layer had a


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thickness of 100-200 µm and bonded well with the titanium mesh (Figure 2d). The bioglass was used to glue the titanium mesh and bioceramic scaffold together after high temperature sintering. The microporosity was helpful for releasing the residual stress resulted from the thermal expansion mismatch. The bioceramic scaffold had macropores and channels with diameters ranging from 100 to 500 µm (Figure 2e and Figure S1), which are ideal for tissue-engineered bone scaffolds to host bone-forming cells, enable nutrient transport, and potentially also allow for vasculature ingrowth

49-51

. As shown in the magnified views in Figure 2f,g, the bioceramic

scaffold was well anchored onto the titanium mesh by the bioglass bonding layer without any interfacial cracking and delamination. The welding strength was 27 ± 0.7 MPa, within the reported bioglass bonding strength range of 20 to 30 MPa 47, indicating that the entire hybrid cage could retain good structural integrity upon loading. EDS elemental mapping and line scanning results suggested that the reaction layer between the titanium mesh and bioglass bonding layer had a thickness of approximately 2 µm and contained the main elements of titanium, silicon, and oxygen (Figure 2g,h and Figure S1). It has been reported that silicon in the bioglass could react 47

with titanium to form Ti5Si3 and TiO2

. Significantly, the material composition, porosity,

dimension, and shape of the hybrid cage are tunable towards resorption rate as various bone sites and geometry requirement for different bone defects. For example, in addition to be used as spine fusion implants, titanium mesh cages have been studied for repair of load-bearing segmental long bone (e.g. femur, tibia) defects

16-20, 52-53

. Therefore the developed manufacturing method may

also be applied to fabricate bioactive titanium-bioceramic hybrid cages for reconstruction of segmentally deficient bones. It should be further noted that this hybrid cage design is materialindependent. Based on the different mechanical and biological requirements for various bone defect sites, the titanium mesh of the hybrid cage can be conveniently replaced by other biometals, such as magnesium, titanium alloys, cobalt-chrome-molybdenum alloys, and stainless steel. The macroporous nWS-HA bioceramic may also be made from other bioceramic systems including


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calcium phosphate, calcium silicate, calcium carbonate, and their composites. With the same design and manufacturing method, we will further be able to fabricate a fully resorbable hybrid cage using biodegradable magnesium and its alloys

54

as the biometal reinforcement for

reconstruction of damaged load-bearing bones in children, which could be resorbed during the growth.

Figure 4 Ion release, biodegradability and biomineralization of the hybrid cage in SBF. The hybrid cage showed excellent biodegradability and rapid biomineralization ability. (a,b) SEM images of various magnifications showing bone-like nanostructured apatite formation on the hybrid cage after immersion in SBF for 1 day. (c) EDS pattern showing the elements within apatite film (black square areas in panel b). (d-f) Phosphorus, calcium, and silicon release profiles (error bars are too small to be visible in these plots). Scale bars: a, 10 µm; b, 200 nm.

Based on the function design of the hybrid cage (Figure 1), the bioceramic scaffold is expected to be replaced by new bone to provide the cage with good bony fusion. At cross-section, the bioceramic scaffold featured an interconnected macroporous structure with pore sizes of 100500 µm while its porosity achieved 85.4 ± 2.6% (Figure 2e,f). It should be noted that the porosity


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and pore size of bone scaffolding material have notable effects on the biological properties including osteoconductivity and bone-forming ability typically results in better osteoconductivity

55-56

28, 49, 51, 55-56

. For example, higher porosity 28

and faster bone regeneration

. Porous

biocoatings on titanium in previous reports have been manufactured with electrodeposition, plasma spraying, electrophoretic coating, slurry dipping and alkali etching

21, 47, 57-59

. Although

these biocoatings achieved considerable porous structure and excellent bonding strength, the limitations could not be disregarded. Still, the porous biocoatings have relatively low porosity/interconnectivity or only small micrometer-sized pores, and are typically too thin to function as engineered scaffolds for optimal bone regeneration. The millimeter-thick bioceramic scaffold structure anchored on our titanium hybrid cage that we have developed in this study on the contrary, had a high porosity of >85% and a pore size of 100-500 µm, which are comparable with optimal requirements for tissue-engineered bone scaffolds (normally having a porosity of around 90% and a pore size ranging from 100-1000 µm 49, 60-61) to allow for transport of nutrients and migration of cells, as well as formation of new bones and vasculature. The manufacturing method could also be used to produce highly interconnected macroporous biocoatings on bone implants, which are hardly achievable using other existing techniques including thermal spray and plasma spray that generate much more glassy phase in the coatings 62. Indeed, the bioceramic scaffold on the hybrid cage showed an interconnected porous structure both on the surface and in the interior (Figure 3a,c), with elongated nWS grains and equiaxed HA grains interlocked together at higher magnifications (Figure 3b,d). The composition of the bioceramic scaffold was analyzed by XRD and EDS (Figure 3e,f), indicating that nWS and HA were stable and co-exsited in the sintered sample. Furthermore, the titanium-bioceramic hybrid cage exhibited excellent biodegradability and biomineralization ability demonstrated by the SBF immersion test and ion release test (Figure 4). SEM images suggested that the hybrid cage possessed a rapid apatite-forming ability (Figure 4a,b). After immersion in SBF for 1 day, a


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uniform nanoporous apatite film was observed to deposit on the surface of the hybrid cage. Figure 4c confirmed the presence of elements of calcium, phosphorus, and oxygen in the apatite films. Figure 4d showed that the phosphorus concentration of the SBF immersed with the hybrid cage dropped (down to 12 µg/mL on day 1) quickly and remarkbly, indicating that the hybrid cage induced rapid apatite formation and biomineralization. As shown in Figure 4e, calcium concentration in SBF immersed with the hybrid cage increased (up to 150 µg/mL on day 1 and 290 µg/mL on day 13) rapidly and dramatically. These results suggested that the hybrid cage had excellent biodegradability and ion release ability due to the anchored bioactive ceramic scaffold. It should be noted that the hybrid cage could also release abundant amount of silicon (Figure 4f), which has been shown to improve that osteoinductivity by promoting osteogenic differentiation of stem cells

32, 34, 63

. The results demonstrated that the hybrid cage possessed excellent

biomineralization capability and biodegradability. The ideal biomaterial for bone repair should interact well with the specific adhesion and growth factor receptors of target cells within surrounding tissues 64. The biomaterial should guide the target cells to migrate into the injury sites and promote their growth

65

. Silicate-based

ceramics including nWS have been reported to possess the ability to induce osteogenesis through inorganic ion release 34. Therefore it is expected that the hybrid spine cage consisting of titanium mesh and nWS-HA boceramic scaffold might be bioactive even in the absence of growth factors. To evaluate their ability to host bone/vessel-forming cells, interact with cells, and promote in

vitro osteogenesis/angiogenesis, three types of cells including MC3T3 preosteoblasts, MSCs, and HUVECs were cultured on the hybrid cage. The live/dead and f-actin/nuclei staining results demonstrated that the hybrid cage could efficiently host the three types of cells to support their attachment, growth, and spreading. The preosteoblasts attached and spread well to form a cellular network on the hybrid cage, which indicated its good biocompatibility (Figure S2). Quantitative analyses revealed high viability of preosteoblasts on the hybrid cage at days 1 and 7 of both >85%


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(Figure S3). Fluorescence microscopy images showed that MSCs with a spindle-like morphology attached and spread on the surface and the interior of the macropores of the bioceramic scaffold on the hybrid cage at day 7, and further proliferated and formed dense cell sheets along the interior of the macropores at day 14 (Figure S4). CLSM images further confirmed the uniform and interconnected distribution of MSCs across the cage through the interconnected macropores (Figure 5a,c), indicating an excellent in vitro bioactivity of the hybrid cage. Quantitative measurement shown in Figure 5e indicated that the hyvrid cage could facilitate proliferation of MSCs. Figure S5 further indicated that MSCs produced biomineralized calcium aggregates on the cage after culturing for 28 days, indicating that it possessed good osteoinductivity to trigger osteogenic differentiation of MSCs to osteoblasts. It can be concluded that the hybrid cage had the ability to host bone-forming cells and exhibited a potential for in vitro osteogenesis. HUVECs were also cultured on the hybrid cage to investigate their in vitro angiogenesis potential. F-actin/nuclei staining further revealed that the HUVECs attached, spread, and proliferated well on the hybrid cage; and by day 14, the HUVECs could form an interconnected cellular network in the macropores (Figure S4). The CLSM images in Figure 5b,d confirmed that the HUVECs could migrate into the inner pores of the bioceramic scaffold on the hybrid cage through the macropores and distribute along the inner surface of the macropores. The quantitative measurement in Figure 5f indicated that the hybrid cage could promote proliferation of HUVECs over the period of culture. It should be noted that one of the greatest challenges in engineering thick and complex tissues (including bones) lies in the need to vascularize the tissue to supply oxygen/nutrients and remove waste products

66

. Our results showed that the interconnected

macropores (100-500 µm in diameter) of the the hybrid cage supported the attachment, spreading, growth, and migration of vascular cells, potentially enabling the formation of a vascular network that is critical for massive bone regeneration 67-68.


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Figure 5. In vitro interaction of MSCs and HUVECs with the hybrid cage on day 14 (phalloidin/DAPI staining for f-actin/cell nuclei). (a,b) CLSM orthogonal views showing the cell morphology and distribution. (c,d) CLSM projection views showing the cell spreading, and cytoskeletal organization. (e,f) Quantification of MSCs and HUVECs proliferations by percentages of fluoresecence-positive area. (* P

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