Effects of dynamic flow rates on degradation deposition behavior of Mg scaffold

Gozhi Ji ,Mng Zhou ,Yiong Hung ,Chnxin Chn ,Ling Jin ,Qin Wu ,Jin Wng,Fi Yu,Ao Xiong,Gungyin Yun,Frnk Fyrbn,Hui Zng,h,f,?

a Department of Bone & Joint Surgery,Peking University Shenzhen Hospital,Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center,Shenzhen Institute of Information Technology,Shenzhen 518036,China

b National Engineering Research Center of Light Alloy Net Forming & State Key Laboratory of Metal Matrix Composite,Shanghai Jiao Tong University,Shanghai 200240,China

c Beijing Key Laboratory of Civil Aircraft Structures and Composite Materials,Beijing Aircraft Technology Research Institute of COMAC,Beijing 102211,China

d College of Life Science,Zhejiang Chinese Medical University,Hangzhou 310053,China

e Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture,Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,China

fDepartment of Orthopaedics,Peking University People’s Hospital,Beijing 100044,China

g Division of Metallic Biomaterials,Institute of Materials Research,Helmholtz-Zentrum Hereon,Geesthacht 21502,Germany

h National & Local Joint Engineering Research Center of Orthopaedic Biomaterials,Peking University Shenzhen Hospital,Shenzhen 518036,China

Abstract Degradability of bone tissue engineering scaffold that matching the regeneration rate could allow a complete replacement of host tissue.However,the porous structure of biodegradable Mg scaffolds certainly generated high specific surface area,and the three-dimensional interconnected pores provided fast pervasive invasion entrance for the corrosive medium,rising concern of the structural integrity during the degradation.To clarify the structural evolution of the three-dimensional (3D) porous structure,semi-static immersion tests were carried out to evaluate the degradation performance in our previous study.Nevertheless,dynamic immersion tests mimicking the in vivo circulatory fluid through the interconnected porous structure have yet been investigated.Moreover,the effects of dynamic flow rates on the degradation deposition behavior of 3D porous Mg scaffolds were rarely reported.In this study,Mg scaffolds degraded at three flow rates exhibited different degradation rates and deposition process.A flow rate of 0.5 mL/min introduced maximum drop of porosity by accumulated deposition products.The deposition products provided limited protection against the degradation process at a flow rate of 1.0 mL/min.The three-dimensional interconnected porous structure of Mg scaffold degraded at 2.0 mL/min well retained after 14 days showing the best interconnectivity resistance to the degradation deposition process.The dynamic immersion tests disclosed the reason for the different degradation rates on account of flow rates,which may bring insight into understanding of varied in vivo degradation rates related to implantation sites.

Keywords: Porous Mg scaffold;Degradability;Porosity;Dynamic immersion test;Degradation rate.

1.Introduction

Tissue engineering provides a promising strategy to promote the regeneration of bone tissue[1,2].Usually,a bone tissue engineering scaffold is designed to mimic the open porous architecture of cancellous bone,bridging the bone defect and function as extracellular matrix [3,4].Ideally,a biodegradable bone tissue engineering scaffold could not only facilitate the tissue growth into the three-dimensional porous structure,but also allow a complete replacement of new bone at controlled degradation rates in line with the tissue regeneration rates[5,6].Mg and Mg alloys have been considered as revolutionary biomedical materials because of the excellent biocompatibility and degradability [7,8].It had been widely reported that in vitro studies of Mg-based biomaterials proved promising osteoconductivity and osteoinductive potential [9,10],the degradation byproduct Mg2+ions could facilitate bone fracture healing by inducing local neuronal production of CGRP[11].Up to now,Mg and Mg alloys have been clinically researched in Germany,South Korea and China in form of bone screws revealing successful treatment [12–14].Thus,the encouraging in vivo and in vitro results indicated that Mg could be advantaged candidate for bone tissue engineering scaffold material.

Mg and Mg scaffolds with various porous structures have been mainly fabricated by mechanical drilling,powder metallurgy,infiltration casting and laser additive manufacturing[15–18].The porous structure certainly generated high specific surface area,and the three-dimensional interconnected pores provided pervasive invasion entrance for surrounding conditions.Then,quick penetration and full contact with the corrosive medium might bring concern of the structural integrity during the degradation of Mg and Mg alloys scaffolds[19].The degradation performance of Mg and Mg alloys were mostly evaluated by electrochemical measurements and immersion tests [20–22].Immersion tests were recommended to investigate the degradation behavior by simulating physiological conditions with simulated body fluid [23,24].Semi-static immersion test employing cell culture conditions and new cell culture medium after certain intervals could partly mimic in vivo renewal of body fluid [25].Retarded degradation rates of Mg alloys were found by applying semi-static immersion tests,the composition of cell culture medium also contributed to the alterable degradation rates [26].However,the in vivo body fluid near the implants,especially,the blood in sites of cancellous bone and compact bone is constantly dynamic[27,28].In addition,the blood flow rates vary dramatically in cancellous bone and compact bone [29,30].It has been reported that the flow rates in the blood vessel system of bone structures are in the range of 0.012–1.67 mL/min[31–33],the normal rate of net filtration in the entire body is 2 mL/min[34].Currently,little is known regarding the influence of flow rates on the degradation behavior of three-dimensional porous Mg scaffolds.

To clarify the effects of dynamic flow rates on degradation deposition process of Mg scaffolds,in this study we carried out dynamic immersion tests under cell culture conditions to investigate the surface and internal degradation morphologies,change of porosities and degradation rates.The degradation deposition behavior together with the interconnectivity of Mg scaffolds were comprehensively evaluated.

2.Materials and methods

2.1. Scaffold preparation

Mg scaffold was prepared by template replication method through infiltration casting process [16].Briefly,irregular polyhedral sodium chloride particles with particle size of about 800 μm were sintered into an open porous template,which followed by infiltrating molten Mg into the pore space.After solidification,the composite of Mg and sodium chloride template was machined into disk-like samples with a diameter of 10 mm and thickness of 2 mm by electrical discharge wire cutting,the sodium chloride particles of the samples were further leached out under tap water.Then,the samples were etched by 1 vol.% nitric acid ethanol within ultrasonic bath for 1 min.Mg scaffolds with irregular polyhedral pores,in the following for simplicity termed I-scaffolds,were received after the etching process.

2.2. Dynamic immersion tests

Dynamic immersion tests were carried out under cell cultural conditions(37°C,5%carbon dioxide,20%oxygen,95%relative humidity).The schematic diagram of the test rig is shown in Fig.1.Two peristaltic pumps(TL15,Medroex,Germany) were installed to maintain medium output and medium input introducing a fluid flow perpendicular to the diameter of porous Mg scaffolds in a PEEK chamber.80 mL of Dulbecco’s modified Eagle medium GlutaMax-1 (DMEM,Gibco,USA) supplemented with 10% fetal bovine serum(FBS,Gibco,USA)and 1%penicillin&streptomycin(Gibco,USA) was recycled for each Mg scaffold.The input medium flow rate at the connection site of scaffold chamber was set to 0.5 mL/min,1.0 mL/min and 2.0 mL/min through the peristaltic pump,respectively.Mg scaffolds were tested at each input flow rate for 14 days,28 days and 42 days,respectively.Four replicates were used for each condition.

Fig.1.Schematic illustration of the immersion test with dynamic flow condition.

2.3. Structural analysis

After the immersion tests,the macromorphology of the degraded Mg scaffolds at the three flow rates were photographed.Scaffolds surfaces were analyzed by scanning electron microscopy (SEM,Tescan VEGA3-SB,Czech Republic) equipped with energy dispersive X-ray spectrometer(EDS).The degraded Mg scaffolds were further scanned using high-resolution micro-computed tomography (μ-CT,Bruker Skyscan 1176,USA) with spatial resolution of 9 μm.3D structural analysis of the reconstructed sample was acquired using the software Mimics 19.0 (materialise,Belgium).The surface area,porosities,volume change of deposition layer and Mg substrate were analyzed by Mimics.

2.4. Degradation rates

The degradation rates of Mg scaffolds at different flow rates were determined by volume-loss measurement.The volume of each scaffold was determined before immersion.The residual volume of Mg substrate was obtained from Micro-CT results.

The degradation rate (mm/year) was deduced according to ASTM G31–72 using the weight difference by the following equation:

WhereKis the coefficient equal to 8.76 × 104,Wis the weight loss (g),Vis the volume loss (cm3),Arepresent the total surface exposed to the medium (cm2),Tmeans the exposure time (h) andDis the density of the material (g/cm3).

2.5. Statistical analysis

All quantitative data were expressed as the means ± standard deviations (n=4 per group,per analysis).The results were analyzed via one-way analysis of variance (ANOVA),andP<0.05 was considered statistically significant.

3.Results and discussion

3.1. Surface degradation deposition morphology

Fig.2 shows the macromorphology of the degraded Iscaffolds at the three different flow rates.A deposition layer was observed under a flow rate of 0.5 mL/min at 14 days,and the porous structure of I-scaffold was severely clogged by the deposition at 42 days.Mg scaffolds degraded at a flow rate of 1.0 mL/min seemed to have more volume lose than those scaffolds degraded at 0.5 mL/min and 2.0 mL/min.A retarded degradation and deposition behavior of Mg scaffolds was revealed at a flow rate of 2.0 mL/min,the hierarchical porous structure of I-scaffold mainly retained after immersion for 28 days.

Fig.2.Optical photographs of the degraded I-scaffolds at three different flow rates.

The surface microstructure of the degraded Mg scaffolds is shown in Fig.3.It indicated that at a flow rate of 0.5 mL/min the deposition products on the irregular polyhedral pore wall of Mg scaffolds consisted of complex morphologies including particles,rodlike and lamellar deposition products,but these products were replaced by clusters of rodlike deposition at 42 days.Mg scaffolds degraded at the flow rate of 1.0 mL/min and 2.0 mL/min for 14 days hardly showed deposition layer on the pore wall.However,after 42 days server degradation and pore strut failure were observed at the flow rate of 1.0 mL/min,while granular deposition with average diameter of about 20 μm was found on the pore wall of Mg scaffolds degraded at 2.0 mL/min.EDS analysis indicated that the granular deposition could be calcium phosphate salts,the clusters of rodlike deposition might be magnesium carbonate,the deposition products formed at the flow rate of 1.0 mL/min could be the mix of magnesium carbonate and calcium phosphate salts.

Fig.3.SEM observation of porous I-scaffolds degraded at three different fluid flow rates,the inserted table is EDS analysis of marked area by square box.

The presented dynamic immersion tests performed a perfusion medium movement by the peristaltic pumps,which achieved a laminar flow through the hierarchical porous structure of I-scaffold.The perfusion fluid supply model was in accordance with the blood transfer feature within cortical bone and cancellous bone [35,36].Thus,the in vitro dynamic immersion tests could mimic in vivo degradation behavior of Mg scaffolds.As the velocity of blood varied due to the physiological structure of host bone [29],effects of flow rates on the degradation morphology of porous structures could further bring insights into biodegradable structural evolution on account of implantation site.According to the surface morphologies after dynamic immersion,the degradation deposition behavior largely depended on the flow rate.Generally,Mg dissolved into solution as Mg2+by the following reactions [37,38].

Together with the dissolution of Mg,the pH value of the solution and the concentration of Mg2+ion near the surface increase,resulted in the formation of degradation deposition products on the surface.Several potential components as degradation deposition products might form on the surface according to the composition of DMEM: Mg(OH)2,MgCO3and Cax(PO4)y[26,39].

The degradation of Mg pore strut might be further facilitated by the attack of Cl-from the corrosive medium according to the following reaction [40].

The degradation byproduct OH-simultaneously reacted with the pH buffer HCO3-of DMEM medium resulting in CO32-production,which subsequently facilitated the deposition of magnesium ion resulting in more magnesium carbonate.The deposition layer of bulk Mg and Mg alloys have been investigated by static immersion tests,whereas the degradation microenvironment could favor the rapid deposition process due to the abundant Mg2+near Mg substrate [41].Our previous study showed that the surface interconnected pores of I-scaffold were clogged by deposition products after 7 days under semi-static immersion conditions [39].In this study,the accumulation of degradation deposition products,especially magnesium carbonate on strut surface at 2.0 mL/min could be significantly reduced by the fast fluid.Meanwhile,the aggressive continuous attack of Cl-might be inhibited because of the dynamic fluid,the initial Mg degradation product Mg(OH)2functioned as protective layer could provide degradation resistance.Moreover,the alkaline environment near pore strut might facilitate the deposition of calcium phosphate salts on Mg surface [42].Thus,a retarded degradation microstructure at 14 days and favorable calcium deposition at 42 days were displayed,respectively.However,Mg2+ion of the microenvironment near strut surface could be accumulated at other two lower flow rates,which would alter the dynamic degradation deposition behavior,the crystallization and growth of carbonate deposition on strut surface could corrupt Mg(OH)2layer resulting in strut failure (1.0 mL/min).Likewise,the deposition products coated the pore strut at 14 days on account of even more Mg2+in pore space (0.5 mL/min),the sustaining high Mg2+concentration could promote the development of magnesium carbonate,which gradually coated the struts and even occupied the pore space.

3.2. Internal degradation deposition morphology

The inspection of internal degraded porous structures is shown in Fig.4.Mg substrate exhibited bright contrast while the deposition products displayed gray contrast due to the relative densities when X-ray penetrated through the degraded scaffolds.The internal porous structures maintained during the whole tests.However,with the accumulated deposition products filled the external pores (0.5 mL/min) and clogged the interconnected pores (1.0 mL/min,2.0 mL/min) after 42 days,the pore space of I-scaffolds decreased,indicating that the interconnectivity was notably inhibited.In addition,the interconnectivity of I-scaffold also depended on the flow rate.The internal porous structure degraded for 28 days at a flow rate of 2.0 mL/min was still well connected,while limited interconnected pores could be identified at other two flow rates.A highly interconnected porous structure of tissue engineering scaffold could facilitate the biological performance by modulating the cell migration,vascularization and transport of nutrients [43].Thus,the maintenance of sufficient interconnectivity is pivotal to the successful bone regeneration [44].The presented Mg scaffolds exhibited fast deposition process resulting in deteriorated interconnectivity at lower dynamic flow rates.However,the degradation deposition behavior of Mg scaffolds could be partly restrained as cell metabolism involved in vivo [45].The protection from cell layer,modulation of proteins and metal ions during cell proliferation and differentiation could be beneficial to degradation resistance sustaining the interconnected porous structures [46].Besides,enlarging interconnected pore size and optimization of strut unit of Mg scaffolds might further enhance the resistance of interconnectivity to the degradation deposition process.

Fig.4.Cross-sectional images of the reconstructed I-scaffolds after dynamic immersion tests.

3.3. Degradation deposition rates

Fig.5A shows the volume evolution of degradation deposition products at the three flow rates during the dynamic immersion tests.The volume of deposition products significantly depended on the flow rate,Mg scaffolds immersed at 0.5 mL/min displayed largest deposition products volume,which was consistent with the SEM observation.Fig.5B shows that the residual volume of Mg substrate decreased with the increment of immersion time.Mg scaffolds degraded at the flow rate of 1.0 mL/min exhibited lowest residual volume,while Mg scaffolds degraded at the flow rate of 2.0 mL/min exhibited much higher residual volume indicating sufficient structural integrity during the dynamic immersion tests.Interestingly,by comparing the deposition products volume with the substrate volume,Mg scaffolds degraded at 0.5 mL/min indicated equal volume at 14 days,nearly one time over the substrate volume after 42 days.Likewise,the substrate volume of Mg scaffolds degraded at 1.0 mL/min and 2.0 mL/min dominated the sample volume.Fig.5C shows that the degradation rates of Mg scaffolds at the three dynamic flow rates,the fluid flow rate 1.0 mL/min induced highest degradation rates,Mg scaffolds immersed at 2.0 mL/min displayed lowest and stabilized degradation rates during the tests.The porosities of the degraded scaffolds are depicted in Fig.5D.With the increment of immersion time the porosities of Mg scaffolds at the three flow rates decreased.Mg scaffolds immersed at 2.0 mL/min showed highest porosity comparing to the other two flow rates,the porosity decreased by about 18% after 42 days.A similar decrement of porosity was found at 0.5 mL/min after 14 days,which further reached to about 38% implying over half of the pore space of Mg scaffolds was clogged by degradation deposition products.

Fig.5.Volume analysis of the degradation deposition layer on the degraded I-scaffolds (A),the residual volume of I-scaffolds (B),degradation rates at the three different flow rates (C) and the change of porosities during the dynamic immersion tests (D).

The degradation deposition behavior of biodegradable Mg scaffolds under dynamic immersion microenvironment illustrated by the change of degradation rates,porosities,most importantly Mg substrate volume and deposition products volume,provided a comparative structural evolution in relation with different fluid flow rates.Collectively,the degradation rates of I-scaffolds at the three flow rates were much lower than those of I-scaffolds degraded at semi-static immersion state in our previous study [39].Semi-static and static immersion tests have been widely used to predict the degradation performance of Mg and Mg alloys,wherein semi-static immersion test employing new cell culture medium after certain intervals in cell incubator might provide comparable physiological conditions with in vivo microenvironment.However,the in vitro degradation rates of Mg and Mg alloys were hardly comparable with in vivo degradation rates due to the complex biological environment of the host [23,38].For example,the in vitro immersion tests rarely mimic the dynamic body fluid.Therefore,the present study of the degradation deposition behavior in consideration of flow rates might bring insight into the mismatched degradation rates between in vivo and in vitro tests.Moreover,according to the dynamic immersion tests on I-scaffold the fluid flow could largely modulate the degradation deposition process within the threedimensional porous structure.The decreased porosities caused by the deposition process evidenced the reduction of the interconnectivity within the degraded Mg scaffolds.In addition,a recent work on porous iron scaffolds revealed that the degradation products could maintain the compressive strength of the degraded scaffold [47].The degradation deposition products of Mg scaffolds might also contribute to sustain the mechanical integrity.A corrosion fatigue test on account of dynamic flow rate would be further conducted in the future work to explore the mechanical integrity of the degraded Mg scaffold.Nevertheless,Mg scaffold degraded at 2.0 mL/min showed a sufficient interconnectivity within 28 days.It could be assumed that under other two lower flow rates Mg scaffolds with enhanced degradation resistance by advanced coating technology could display a comparable porous structural integrity with the bare Mg scaffolds degraded at 2.0 mL/min,or even better retained interconnectivity.

4.Conclusions

In the present study,the comprehensive effects of flow rates on the degradation deposition of Mg scaffolds were investigated by dynamic immersion tests.The results revealed that changing the flow rate could modulate the degradation deposition morphologies of surface and internal microstructure.A fast deposition layer was found at a flow rate of 0.5 mL/min after immersion for 14 days and clogged the external porous structure after 42 days,while the interconnected porous structures of Mg scaffold partly retained at 2.0 mL/min after 42 days.The deposition of degradation byproducts contributed to the decrease of porosities,which simultaneously deteriorate the interconnectivity of the whole scaffold.The degradation byproducts at 0.5 mL/min occupied half of the pore space after 14 days,indicating excessive deposition rates.The flow rate 1.0 mL/min generated highest degradation rate implying insufficient protection of deposition layer.However,Mg scaffolds degraded at the three flow rates showed significant lower degradation rates in comparison with the Mg scaffolds degraded at semi-static immersion tests in our previous study.To maintain an interconnected porous structure comparable with the scaffolds degraded at 2.0 mL/min and enhance the structural integrity,coating technology would be prerequisite for in vivo study.Additionally,optimization of pore strut unit could be another option to reduce the specific surface area and avoid the accumulation of degradation byproducts.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by grants from National&Local Joint Engineering Research Center of Orthopaedic Biomaterials (XMHT20190204007),Shenzhen Key Medical Discipline Construction Fund (No.SZXK023),Shenzhen “San-Ming”Project of Medicine (No.SZSM201612092),Shenzhen Research and Development Project (No.Z2021N054),Guangdong Basic and Applied Basic Research Foundations (No.2019A1515011290,2021A1515012586,2019A1515110983),China Postdoctoral Science Foundation (No.2020M672756),Bethune Charitable Foundation and CSPC Osteoporosis Research Project (No.G-X-2020–1107–21).The authors would like to acknowledge the introduction from Dr.Jorge Gonzales for the dynamic immersion test.