Copper-modified Ti6Al4V suppresses inflammatory response and

Subscriber access provided by Uppsala universitetsbibliotek

Tissue Engineering and Regenerative Medicine

Copper-modified Ti6Al4V suppresses inflammatory response and osteoclastogenesis while enhancing extracellular matrix formation for osteoporotic bone regeneration Xiongcheng Xu, Yanjin Lu, Xue Yang, Zhibin Du, Ling Zhou, Shuman Li, Chao Chen, Kai Luo, and Jin-xin Lin ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00736 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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

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

Page 1 of 46 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

ACS Biomaterials Science & Engineering

Abstract: Copper has been reported to promote bone regeneration by increasing osteogenesis and decreasing inflammation and osteoclastogenesis. However, information on the effects of copper on osteoporotic cells involved in bone regeneration is scarce in the literature. In the current study, Ti6Al4V-6wt.%Cu (Ti6Al4V-Cu) was fabricated by selective laser melting (SLM) technology, and the effects of copper on the behaviors of osteoporotic and non-osteoporotic macrophages, osteoclasts and osteoblasts were evaluated by comparison with Ti6Al4V. Our results showed that Ti6Al4V-Cu inhibited the activation, viability and pro-inflammatory cytokine secretion of osteoporotic macrophages and decreased osteoclast formation and down-regulated osteoclast differentiation-related genes and proteins of osteoporotic osteoclasts. Furthermore, the bone extracellular matrix formation of osteoporotic osteoblasts was up-regulated by Ti6Al4V-Cu. In conclusion, SLM-fabricated Ti6Al4V-Cu exhibited excellent anti-inflammation and anti-osteoclast capability, optimized extracellular matrix formation, and hold great potential for bone regeneration in osteoporotic patients. Keywords: Copper; selective laser melting; osteoporosis; anti-inflammation; osteoclastogenesis; extracellular matrix

Introduction Periodontitis is a biofilm-induced chronic inflammatory disease that destroys periodontal support tissue, such as the underlying alveolar bone 1. Simultaneously with the acceleration of the aging process, postmenopausal osteoporosis (PMO) has reached notably high rates and could cause an imbalance in alveolar bone homeostasis, ultimately leading to a reduction of bone mass 2. Consequently, bone regeneration of periodontitis patients with osteoporosis is a great challenge.

2

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

Guided bone regeneration (GBR) supplies spaces for various cells involved in bone regeneration, such as osteoblasts, osteoclasts and macrophages, and prevents soft tissue in-growth using barrier membranes 3. In clinical practice, the titanium mesh membrane has been extensively used in GBR due to its rigidity, which avoids contour collapse and allows extensive space 4. However, titanium mesh membranes are indispensable to bend and contour to adapt to the unique bone defects during GBR surgery, which takes time and requires experienced operation 5. Furthermore, the bone sites of GBR experience constant microbial challenge in the oral cavity, which increases the risk of infection, especially after titanium mesh membrane exposure, and might ultimately result in bone regeneration failure. In addition, the titanium mesh membrane in current clinical use acts only as a barrier and has no direct effect on regulation of cells participating in bone regeneration, the curative effects of which are poor for patients with low bone regenerative capacity, such as osteoporotic patients.

Recently, selective laser melting (SLM) technology based on three-dimensional (3D) model data has been proven as an appropriate approach that can produce patient-individual materials quickly and cost-efficiently 6-7, and customized Ti6Al4V implants fabricated by SLM have been applied in surgical reconstruction 8. For patients undergoing GBR surgery, custom-designed titanium mesh membranes could be fabricated by SLM, according to the different bone defects, to recover the anatomical morphology and avoid pre-bending of the titanium mesh membrane during surgery. Copper ions have a brilliant antibacterial property against both Gram-positive and Gram-negative bacteria 9. Ren et al. and Ma et al. incorporated copper ions into Ti6Al4V and demonstrated that the copper-bearing alloy fabricated by powder metallurgy possessed an obvious antibacterial effect due to the release of copper ions

10-12

, which was also observed in copper-coated Ti6Al4V and copper-bearing alloys fabricated by

laser powder bed fusion

13-14

. Additionally, certain research studies demonstrated that copper ions not

3


Page 2 of 46

Page 3 of 46 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


only inhibited the inflammatory response and osteoclastogenesis and angiogenesis

17-18

15-16

but also promoted osteogenesis

. Prinz et al. found that copper-coated Ti6Al4V nails enabled the stimulation of

bone formation in a rabbit model 19. Furthermore, copper deficiency is associated with osteoporosis and low bone density, and supplementation with copper is recommended 20. The above information implies that remarkable potential exists for copper ions applied to the barrier in GBR for osteoporotic patients. Our previous study successfully fabricated a group of copper-modified Ti6Al4V alloys with different copper levels using SLM and found that the Ti6Al4V-Cu alloy composed of 6% copper (by weight) released copper ions in a sustained manner and displayed a strong antibacterial property and excellent biocompatibility

21-22

. However, the effects of Ti6Al4V-Cu alloy with 6 wt% copper in

practical applications are still unknown, especially its impacts on osteoporotic cells involved in bone regeneration. Therefore, our current research focused on the basic effects of Ti6Al4V-Cu on osteoporotic macrophages, osteoclasts and osteoblasts to elucidate the potential mechanism and offer a theoretical basis for prospective clinical transformation. Materials and methods Preparation of SLM-fabricated Ti6Al4V-Cu alloy The Ti6Al4V-Cu alloy was prepared as described in the literature 22. In brief, commercial Ti6Al4V and pure copper powders were used as the raw materials in the SLM with a cross-hatching technique on an SLM machine (Mlab-R, Concept Laser GmbH, Germany). The laser power was set to the highest value of 95 W, and a laser scanning speed of 900 mm/s, layer thickness of 25 µm and track width of 77 µm were selected to fabricate the alloys. The linear scanning strategy was used, and the laser scanning pattern between layers was rotated by 90° (cross-hatching technique). To remove the residual stress of the alloys, all specimens were heated to 875 °C and held for 2 h, followed by water quenching. The

4



Ti6Al4V-Cu alloy contained 6% copper (weight percentage). As a control group, the Ti6Al4V alloy was fabricated by applying the same processing method without the addition of copper powder. All samples measuring 10×10×3 mm were used in the cell experiments. Before the start of the experiments, the samples were prepared by grinding with SiC papers of 360, 1000 and 2000 grit sizes, polished using a SiO2-H2O2 solution (Buehler, USA), cleaned with acetone and 75% ethanol via ultrasonication for approximately 30 min, and dried at room temperature. Analysis of copper ion release and mechanical property of Ti6Al4V-Cu According to the international standard ISO 10993-12, Ti6Al4V-Cu was immersed in Dulbecco’s modified Eagle medium (DMEM) (HyClone, USA) with a surface-area-to-volume ratio of 3 cm2/mL under the conditions of 37 °C and 5% CO2 in air for 1, 3, 5, 7 and 9 days to prepare the extracts (n=3 at each time point)

23

. The concentration of released copper ions was detected by inductively coupled

plasma-atomic emission spectroscopy (ICP-AES, Ultima2). The tensile test of Ti6Al4V-Cu was evaluated according to ASTM E8/E8M. The tensile specimen was fabricated by SLM and its schematic diagram of tensile specimen is shown in Figure S1a. Tensile tests were performed using an MTS criterion universal testing system (MTS Corporation, USA) at room temperature, with an initial strain rate of 2 mm/s. Cell culture from osteoporotic rats All study protocols and animal care procedures were approved by the Animal Care and Use Committee of Fujian Medical University. A rat model of osteoporosis was established via bilateral ovariectomy, as previously described

24

. In brief, three-month-old female Sprague-Dawley rats

weighing 250-280 g were obtained from the animal resource center (SLAC Laboratory Animal Co, Ltd, Shanghai, China). After 1 week of adaptive feeding, 20 rats were chosen randomly and ovariectomized

5


Page 4 of 46

Page 5 of 46 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


bilaterally (OVX group), and an equal volume of fat tissue was excised from a location next to each ovary of the other 10 rats (control group). At 12 weeks after the operation, bone marrow-derived macrophages were isolated from the femurs of the control group and OVX group, as described in the literature

25

. In brief, bone marrow was

washed from the femur with DMEM and cultured in 75-cm2 flasks (Thermo Fisher Scientific Inc., Pittsburgh, USA) in a medium consisting of 10% fetal bovine serum (FBS) (HyClone, USA), 90% DMEM and 10 ng/mL of rat recombinant macrophage-colony stimulating factor (M-CSF) (Peprotech, Rocky Hill, NJ, USA). The bone marrow-derived macrophages were also used as osteoclast precursor cells and treated with 25 µg/l M-CSF and 45 µg/l receptor activator for nuclear factor-κ B ligand (RANKL) (Peprotech, Rocky Hill, NJ, USA) to induce osteoclasts, as described in the literature26. Osteoblasts were isolated from the control and OVX groups, as described in the literature27. In brief, the mandibles of osteoporotic rats were removed under sterile conditions and cut into bone slices. Osteoblasts were isolated by incubation with trypsin and type II collagenase. All cells were cultured in a humidified environment. The medium was changed every 2-3 days. The cells were passaged when they became 70-80% confluent. Macrophages were cultured on Ti6Al4V and Ti6Al4V-Cu alloys placed in 24-well culture plates (Thermo Fisher Scientific Inc., USA) at an initial density of approximately 1 × 105 cells per well, and the density of osteoblasts was 2 × 104 cells per well at 37 °C in a humidified atmosphere containing 5% CO2. The samples were brought to new 24-well culture plates at different testing time points to conduct following experiments. Cell morphology and immunofluorescent staining To evaluate the attachment and morphology of cells on Ti6Al4V and Ti6Al4V-Cu alloys, the cell/material constructs (n = 6) were removed from the culture wells at different time points of culture,

6



Page 6 of 46

rinsed with phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde for 20 min. Excess paraformaldehyde was removed by washing with PBS. The cell/material constructs were permeabilized in 0.1% Triton-X-100 in PBS and blocked with 2% bovine serum albumin (BSA) for 1 h at room temperature. Subsequently, the cells were incubated with rabbit monoclonal antibody against C-C chemokine receptor 7 (CCR7) (1:100, Abcam, USA), mannose receptor C-type 1 (MRC1) (1:200, Abcam, USA), tartrate resistant acid phosphatase (TRAP) (1:100, Abcam, USA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (1:300, Abcam, USA), collagen type-1α1 (Col-1α1) (1:500, Abcam, USA) and lysyl oxidase (LOX) (1:300, Abcam, USA) for 2 h at room temperature. Fluorescent labeling with secondary antibodies was performed using Alexa Fluor 488 goat anti-rabbit (1:1000, Invitrogen, Carlsbad, CA, USA). The actin cytoskeleton was stained with rhodamine phalloidin (stock solution in methanol diluted to 1:1000, Cytoskeleton Inc., USA). The 4’,6-diamidino-2-phenylindole (DAPI) solution (5 mg/mL) was added to counterstain the cell nuclei. The stained cells were imaged using fluorescence microscopy (Olympus, Japan). The macrophage spread area and perimeter on Ti6Al4V and Ti6Al4V-Cu were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) based on cytoskeleton staining. The macrophage circularity was also assessed. Cell Circularity = (4π × area)/(perimeter)

2

and for a perfect circle is

equal to 1, and as the shape becomes more convoluted, the value decreases and is

Copper-modified Ti6Al4V suppresses inflammatory response and

Recommend Documents