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[Nur Rofiqoh Eviana Putri](https://orcid.org/0000-0002-4492-4956), Huajian Chen, [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), [Felicity R. A. J. Rose](https://orcid.org/0000-0001-6640-8840), Ricky D. Wildman, [Guoping Chen](https://orcid.org/0000-0001-6753-3678)

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[Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering](https://mdr.nims.go.jp/datasets/839ff0f8-b0db-4502-a3c3-4c90c4f3090e)

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Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineeringRSC AdvancesPAPEROpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View IssueTailoring cell behaDepartment of Chemical Engineering, FacMada, Jl. Graka No 2, Kampus UGM, Yoroqoh.e@ugm.ac.idbResearch Center for Macromolecules anMaterials Science, 1-1 Namiki, Tsukuba, IbCHEN@nims.go.jpcBiodiscovery Institute, School of PharmacyNG7 2RD, UKdCentre for Additive Manufacturing, FacultyNottingham, NG7 2RD, UKCite this: RSC Adv., 2025, 15, 28581Received 25th April 2025Accepted 1st August 2025DOI: 10.1039/d5ra02891drsc.li/rsc-advances© 2025 The Author(s). Published byaviour by surface micropatterningand interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bonetissue engineeringNur Rofiqoh Eviana Putri, *a Huajian Chen,b Naoki Kawazoe,bFelicity R. A. J. Rose, c Ricky D. Wildmand and Guoping Chen *bScaffold architecture with complementary features on the surface brings the desired properties in thesurface chemistry. That structure plays a critical role in tissue engineering to tailor cell behaviour andpromote effective transport for cell growth and tissue regeneration. In this work, a controllableinterconnected three-dimensional (3D) porous scaffold with surface micropatterning was fabricated.Nozzle-based Aerojet dispenser 3D printing was used to form printed ice as a fugitive ink combined witha freeze-drying method of gelatin/nano-silica/poly lactic-co-glycolic acid (PLGA) and ice particulates tofabricate a composite scaffold with supporting properties. Several designs of printed ice were exploredand the HUVECs' behavior on different surface patterns was analyzed. The results showed that HUVECsexhibited orientation adhesion and growth with a certain direction after 6 days of culture. The 3D-controlled interconnected porous scaffolds with surface micropatterning then were used for the 3Dculture of hMSCs. The hMSCs analysis showed a facilitating effect for cell distribution and growth in the3D composite scaffolds compared to the control scaffold without interconnected porous structure andsurface micropatterning. This study demonstrated that controlled cell behavior by patterning the surfaceof the scaffold and improved cell growth by controlling the interconnected inner porous scaffold hasa significant role in bone tissue engineering.1 IntroductionBone regeneration is a very complex process that needs twocrucial factors, i.e. blood supply to the defect and xationstability (biomechanics).1 It follows the bone healing cascadeincluding the inammatory (hematoma formation to promoteangiogenesis), reparative (bone callus formation), and boneremodeling process.2,3 The bone regeneration cascade is usefulfor small defects (less than 2 cm) because it is a highly vasculartissue. In a large defect, it is still difficult for the bone tissue torepair and regenerate by itself. That leads to permanent bonedefects and loss of function.4 Therefore, clinical treatment isneeded for further regeneration to initiate the inammatoryulty of Engineering, Universitas Gadjahgyakarta, 55284, Indonesia. E-mail: nur.d Biomaterials, National Institute foraraki, 305-0044, Japan. E-mail: Guoping., University of Nottingham, Nottingham,of Engineering, University of Nottingham,the Royal Society of Chemistrysteps in the initial bone regeneration cascade due to the lack offeatures for the transport process.The eld of tissue engineering, which focuses on regene-rating tissue defects by using a combination of cells, growthfactors, and scaffolds offers great advantages for bone treat-ment.5 Tailoring the scaffold architecture with supportingfeatures is essential to promote the transport of essentialmolecules (oxygen, glucose, and amino acids) and eliminate theby-products of the degradation process. Cells within the bodyare located in no more than 100 mm of vascular channels.6,7Without the interconnected porous structure, nutrient trans-port is dependent only on diffusion, which is only efficient forshort depths. Thus, in large scaffolds, the penetration ofnutrients occurs on the surface, leading to cell death at thehypoxic core. It triggers the inammatory responses and rejec-tion of the native tissues and cannot form an excellent inte-gration with the host tissue.8Besides the interconnected pores inside the bulk of thescaffold, the surface properties also perform a critical role in thecell attachments, migration, and proliferation as well as theadsorption of bioactive molecules, which leads to successfultissue regeneration.9,10 Several strategies have been developed tocreate a scaffold with surface patterning such as lyophilizationRSC Adv., 2025, 15, 28581–28591 | 28581http://crossmark.crossref.org/dialog/?doi=10.1039/d5ra02891d&domain=pdf&date_stamp=2025-08-11http://orcid.org/0000-0002-4492-4956http://orcid.org/0000-0001-6640-8840http://orcid.org/0000-0001-6753-3678http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dhttps://pubs.rsc.org/en/journals/journal/RAhttps://pubs.rsc.org/en/journals/journal/RA?issueid=RA015035RSC Advances PaperOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlineand molding techniques,10 phase separation micro-moldingprocess,11 electrospinning and molding techniques,12 and elec-tron beam lithography.13 They can align customized networksand patterning using a broad range of materials, simpleprocesses, and low cost. However, they have limitations inachieving a complex structure, especially for 3D constructfabrication.Previously developed scaffolds were prepared by combinedthe surface patterning and porous bulk scaffold to face thosechallenges. The work by Guicai Li, et al. demonstrated thefabrication of a micropatterned scaffold to guide the endothe-lial cell alignment by using a PDMS stamp for casting the chi-tosan surface patterning and lyophilization to form the porousstructure on the bulk of the scaffold. However, it lackedcontrolled internal porosity, which is essential for effectivenutrient diffusion.14 Additionally, Maria Moffa, et al. presentedcombined micro- and nano-topographic cues including thegrooved micropatterns and electrospun bers to guide theendothelial cell alignment. However, their approach lackedvariation in the surface patterns.12In order to solve those problems, this work combined the useof additive manufacturing also known as 3D printing with theice particulates/freeze drying method to form a 3D scaffold withcontrolled interconnected inner porous structures and surfacemicropatterning. The surface pattern of the scaffold couldregulate the endothelial cell behaviour, meanwhile the inter-connected pores on the bulk of the scaffold could enhancenutrient transport and cell penetration. In order to forma micropatterned surface scaffold, Aerojet dispenser 3Dprinting was used to print water onto a frozen substrate with theprepared design. The Aerojet dispenser can offer great exibilityfor more complex pattern formation with biocompatibility.Shangwu Chen, et al. used the Aerojet dispenser for micro-grooved collagen porous scaffold fabrication to engineer skel-etal muscle tissue.15 They successfully controlled the alignmentof myoblasts, which resulted in the synthesis of muscle extra-cellular matrix. However, they have not explored differentpatterns besides the microgroove yet and did not control theinterconnected porous scaffolds. Another study was conductedby Ying Chen, et al. by fabricating the composite scaffold ofdexamethasone-loaded calcium phosphate nanoparticle/collagen with a microgroove network.16 They presentedpromotive effects for angiogenesis and osteogenesis on themicro-grooved composite scaffold compared to the controlscaffold. However, they have not studied the different patternsthat potentially affect the HUVECs' alignment and orientation.Therefore, in this work, we explored the formation of differentpatterning and optimized the parameters during printing toform the high-resolution and stable printed ice.Bioactive silica is selected as one of the materials for scaffoldfabrication since provides biomineralization capability.17 Theuse of nano-silica for scaffolds increased protein adsorptionand controlled swelling ability. It was also biodegradable andimproved apatite deposition.18 However, silica scaffolds havelow formability and high brittleness, so gelatin and PLGA wereadded to improve the stiffness and mechanical properties.19 Inaddition, gelatin has a similar composition to collagen and28582 | RSC Adv., 2025, 15, 28581–28591excellent biocompatibility, which is oen used for the regen-eration of bones and cartilage.20 Meanwhile, the PLGA hascontrollable mechanical properties, which could improve thestrength and stability of the composite material.21 Finally, the3D construct of scaffolds with surface patterned and controlledinterconnected porous structures of gelatin/nano-silica/PLGAcomposite scaffolds were explored.2 Materials and methods2.1 Exploration of 3D-printed iceFrozen 3D-printed ice was used as sacricial ink to formdifferent patterns on the surface of the scaffold. The 3D printingof water onto frozen substrate was done using a jet dispenser(MJEY-3-CTR, Musashi Engineering Inc), which was controlledby a SHOT mini 200a (Musashi Engineering Inc). A differentpattern was prepared by inputting the program on the SHOTmini 200a to control the movement of X, Y, and Z directions.Peruoro alkoxy lm (PFA lm, Universal Co., Ltd) was wrappedon a copper plate, which was then frozen by liquid nitrogen.Ultrapure water was lled on the syringe and ejected througha 0.140 mm nozzle by applying the air pressure, that was con-nected to the jet dispenser. The water droplets were immedi-ately frozen and formed the pre-prepared pattern of frozenprinted ice.The xed parameters during printing were a printing speedof 40 mm s−1, on time (time of water being held at the edge ofthe nozzle) of 3 ms, number of layers of 4, and a nozzle diameterof 0.14mm.Meanwhile, some parameters were varied includingthe off time (interval time between the water droplet), and airpressure. The printed line pattern was captured using anOlympus microscope with a DP22 camera so that the line widthand line gap of the printed line can be measured using ImageJsoware (ImageJ2, NIH) on 4 images of each varied parameter.2.2 Fabrication of porous gelatin scaffold with differentsurface patternsA porous scaffold with a different surface pattern (Scheme 1(A))was prepared by using the printed ice to control the surfacepattern and a mixture of gelatin with ice particulates to form theporous scaffold aer the freeze-drying process. The printed icewas prepared as described before using optimum printingconditions, i.e., the printing speed of 40 mm s−1, on time of 3ms, nozzle diameter of 0.14mm, off time of 9 ms, air pressure of0.006MPa, and number of layers of 4. On the other hand, the iceparticulates were prepared by spraying Milli-Q water into liquidnitrogen using a sprayer. They were then sieved by sieves withmesh pores sizes of 150 and 250 mm to control the diameter ofice particulates. The sieving process was performed in the low-temperature chamber (ESPEC, Osaka, Japan) at −15 °C.Gelatin solution at 4.0 (w/v)% was prepared by adding thegelatin granules (porcine-derived gelatin, Nitta Gelatin, Inc.)into 30.0 (v/v)% acetic acid as the solvent. It was stirred at 45 °Cfor 2 hours and then continued stirring at room temperature for1 hour. The acetic acid solution was used as a solvent to reducethe gelation of the gelatin solution at low temperatures. All pre-© 2025 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dScheme 1 Schematic preparation of (A) scaffold with different surfacepatterns, and (B) 3D sandwich scaffold with patterned surface andinterconnected porous structure.Paper RSC AdvancesOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlineprepared ice particulates, frozen printed ice, gelatin, and toolswere kept at −5 °C for 2 hours to allow temperature balanceprior to assembly.The ice particulates were added into 4.0 (w/v)% gelatinsolution with the ratio of ice particulates/gelatin solution of 70 :30 (w/v) and thoroughly mixed until homogeneous. In order tocontrol the thickness of the scaffold, a 1mm thick siliconemoldwas placed onto frozen printed ice. The mixture of iceparticulates/gelatin was then poured onto the silicone mold,attened with a steel spatula and then covered with a glassplate. It was moved and kept at −80 °C freezer for at least 6hours to ensure complete freezing. The frozen construct wasdetached from the glass plate and then freeze-dried for 48hours.2.3 Fabrication of gelatin/nano-silica/PLGA 3D compositescaffolds with patterned surface and interconnected porousstructureScaffolds were prepared by frozen printed ice, ice particulates,vicryl knitted mesh of polyactin 910 (a 90 : 10 copolymer ofglycolic acid and lactic acid (PLGA) Ethicon, Inc., Somerville, NJ)mesh, nano-silica, and gelatin solution. The schematic fabri-cation is shown in Scheme 1(B). First, an 8.0 (w/v)% gelatinsolution was added to a 2.0 (v/v)% nano-silica suspension toprepare a gelatin/nano-silica mixture with the nal concentra-tion of 4.0 (w/v)% gelatin and 1.0 (v/v)% nano-silica in 35.0 (v/v)% acetic acid. It was stirred at room temperature for 1 hour toform a homogeneous mixture. The materials and tools werethen kept in a −5 °C low-temperature chamber for 2 hours tobalance the temperature. Subsequently, a 1 mm thick siliconemold was placed onto frozen printed ice, which was preparedabove the PFA-wrapped copper plate. A mixture of iceparticulates/gelatin/nano-silica with a ratio of ice particulatesand gelatin/nano-silica mixture of 70 : 30 (w/v) was poured onto© 2025 The Author(s). Published by the Royal Society of Chemistryfrozen printed ice with the silicone mold. The surface of themixture was attened with a steel spatula and covered witha knitted PLGA mesh, which was then immersed in a gelatinsolution. Another 1 mm thick silicone mold was placed onto theconstruct and then poured with another mixture of iceparticulates/gelatin/nano-silica. The surface of the mixture wasattened with a spatula and covered with frozen printed ice.Aer mixing, the construct was moved into a−80 °C freezer andkept for at least 6 hours to ensure complete freezing then freeze-dried for 48 hours. As a control, a scaffold was prepared withoutthe frozen printed ice on the surface, nano-silica, and iceparticulates on the matrix.2.4 Crosslinking of scaffoldsChemical crosslinking using 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Peptide Institute,Inc), 20 mM N-hydroxysuccinimide (NHS, Wako Pure ChemicalIndustries, Ltd) and 0.1(w/v)% 2-(N-morpholino) ethanesulfonicacid (MES) in three solvents of decreasing ethanol concentra-tions with ratio of ethanol/water of 95/5, 90/10 and 85/15 (v/v).Aer the freeze drying, the scaffold was immersed in pureethanol by degassing, followed by soaking in the rst cross-linking solution (ethanol/water 95/5) for 8 hours, the secondsolution (ethanol/water 90/10) for 8 hours, and then the thirdsolution (ethanol/water 85/15) for 8 hours. To remove theunreacted reagents, the scaffold was then rinsed with MiliQwater 10 times at room temperature and re-freeze dried for 48hours to get the nal crosslinked scaffold.2.5 Characterization of scaffoldsThe morphology of scaffolds was analyzed by a JSM-5610Scanning Electron Microscope (SEM; JEOL, Tokyo, Japan). Thesurface, horizontal, and vertical cross-sections of scaffolds weresputter-coated with gold before characterization. Samples wereobserved at a 10 kV acceleration voltage and the pore size of thescaffold was calculated using four SEM images of each type ofscaffold by measuring the diameters of pores using ImageJsoware (ImageJ2, NIH).For porosity measurement, the scaffolds were punched usinga biopsy punch with a diameter size of 6 mm. The calculationwas conducted by measuring the weight of water lling the voidspaces in the scaffolds using the following equations:22,23Porosity ¼ VporeV� 100% ¼ W2 �W1rV� 100% (1)where Vpore is the pores volume, V is the scaffold volume, W1 isthe weight of the dry scaffold, W2 is the weight of the hydratedscaffold with water, and r is the density of water. Triplicatesamples were used for the measurements.2.6 In vitro cell culture of HUVECs onto different surfacepatterns of porous scaffoldHUVECs (C2519A, Lonza) were used for the in vitro cell culture onthe scaffolds with different surface patterns. Before cell seeding,the scaffolds were punched into cylindrical discs (F10 mm×H 1mm) and then sterilized by immersing them in ethanol for 1RSC Adv., 2025, 15, 28581–28591 | 28583http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dScheme 2 Schematic in vitro cell culture of (A) HUVECs on thescaffold with different surface patterns, and (B) hMSC on the 3Dsandwich composite scaffold with patterned surface and inter-connected porous structure.RSC Advances PaperOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehour. They were washed with PBS 5 times and then immersed inDMEM medium for at least 2 hours. The HUVECs were sub-cultured using endothelial cell growth medium (EGM2, CC-3162, Lonza) that contained EGM2 Single Quots (CC-4176,Lonza). Aer reaching conuence, the cells were harvestedusing trypsin/EDTA and then re-suspended in DMEM to preparea cell suspension solution with the density of 2× 106 cells per mLfor cell seeding. 90 mL of the HUVECs suspension solution wasdropped on the sterilized scaffold and incubated for 3 hours toprovide the cells attachment. Aer 3 hours, the glass ring witha diameter of 10mm, which was placed on the scaffold to providethe cell seeding was removed, and then DMEM low glucosemedium supplemented with 10 mM b-glycerophosphate and100 nM dexamethasone (Dex) was added into scaffold disc. Themedium also contained 10% fetal bovine serum, 4 mM gluta-mine, 100 U ml−1 penicillin, 100 mg ml−1 streptomycin, 0.1 mMnonessential amino acids, 0.4 mM proline, 1 mM sodium pyru-vate and 50 mg ml−1 ascorbic acid. Seeded samples were incu-bated in a humidied incubator at 37 °C and 5% CO2 with themedium being changed every 3 days (Scheme 2(A)).2.7 In vitro cell culture of hMSCs onto a 3D compositescaffold with patterned surface and interconnected porousstructure3D composite scaffolds were punched into cylindrical discs witha diameter of 6 mm and then sterilized by immersion in ethanolfollowed by washing with PBS 5 times. The hMSCs (passage 4,Lonza) were sub-cultured using MSCBM medium (Lonza) untilreached conuence. They were then harvested using trypsin/EDTA and re-suspended in DMEM to obtain a cell suspensionsolution of 2 × 106 cells per mL. 80 ml of hMSCs suspensionsolution was seeded onto one side of the scaffolds and culturedat 37 °C for 3 h. Aer that, the scaffolds were turned upsidedown and another 80 ml of hMSCs suspension solution wasseeded. They were then cultured at 37 °C for 3 h to allow cellattachment and fresh culture medium was added. Aer 1 day,the seeded scaffolds were moved to a 75 cm2 tissue culture askand cultured under shaking at 60 rpm with the medium beingchanged every 3 days (Scheme 2(B)).2.8 Cell characterizationAer 1 day of culture, cell adhesion and distribution in the 3Dcomposite sandwich scaffolds were analyzed by SEM. Sampleswere rinsed with PBS and xed with 2.5% glutaraldehyde at roomtemperature for 1 hour. Aer that, they were washedwith PBS andwater respectively, and were freeze-dried for 48 hours. Before theanalysis, the surface, horizontal cross-section, and vertical cross-section were spin-coated with gold and observed by SEM.Hoechst 33 528 staining was used to observe the cells nuclei ofthe seeded cells on the scaffolds aer 6 days of culture. Sampleswere washed with PBS and xed in a 4% paraformaldehyde for 30minutes at room temperature. Aer that, they were permeabilizedwith 0.2% (v/v) Triton X-100 for 15 minutes, followed by washingwith PBS. 2 mg mL−1 Hoechst 33 528 (343-07961, Dojindo) wasadded into the xed sample and kept for 15 minutes at roomtemperature. For staining of F-actin, Alexa Fluor 488 Phalloidin28584 | RSC Adv., 2025, 15, 28581–28591(Life Technologies) was diluted in PBS with a ratio of 1 : 40 andincubated for 40minutes. During the incubation, samples in wellplates were covered with aluminium foil to avoid light. Thestained samples were washed with PBS and observed by uores-cence microscope (Olympus, Japan).Life/dead staining was used to check the viability of the cellsaer 1, 4, and 6 days of culture using calcein-AM and propidiumiodide staining reagents (Cell-stain Double Staining Kit,Dojindo, Japan). Samples were washed with PBS and incubatedin the staining solution for 15 minutes. During the incubation,samples in well plates were covered with aluminum foil to avoidlight. Stained samples were analyzed by uorescence micro-scope (Olympus, Japan).Alizarin red S and alkaline phosphatase (ALP) staining wereperformed on the hMSCs seeded on the scaffold aer 7 days ofculture. Samples were washed with PBS three times beforebeing xed in the 4% paraformaldehyde for 10 minutes at roomtemperature. Aerward, xed scaffolds were washed with PBStwo times and then stained with the staining solution for 10minutes at room temperature. During the staining process, thesample well plates were covered with aluminum foil to avoid theinterference of the light. The staining solution of ALP contains0.1% fast blue RR salt and 0.1% naphthol AS-MX phosphate,which is diluted in 56 mM 2-amino-2-methyl-1,3-propanediolsolution (pH = 9.9). Meanwhile, the alizarin red S stainingsolution contains 0.5% alizarin red S in PBS solution. Thestained samples were then washed with PBS three times andsectioned with a blade to obtain the vertical cross-section. Anoptimal microscope was used to observe the sectioned samples.3 Results and discussion3.1 Exploration of 3D-printed iceThe Aerojet dispenser 3D printer, which utilizes air pressure toeject water through a nozzle and form a different pattern on the© 2025 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dFig. 1 Formation of 3D printed ice on a frozen copper plate coveredby PFA film at different printing settings.Fig. 2 Formation of printed ice at different designs of pattern on top ofthe frozen copper plate (top row) and porous gelatin scaffold atdifferent designs of a surface pattern after freeze-drying (bottom row).Paper RSC AdvancesOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinesurface of the scaffolds was used. The resolution of the Aerojet3D printing is essential to form the pattern precisely. Therefore,some parameters during printing including the off time (timeinterval between the jetting of two water droplets) and airpressure were explored and controlled to obtain a high resolu-tion and stable jetting of ultra-pure water forming 3D frozenprinted ice as shown in Fig. 1.Ultrapure waters were dropped onto a frozen copper plate,which was covered by hydrophobic polyuoroalkoxy (PFA) lm.The use of PFA lm was important to provide a good surfacetension between water and surface, thus forming a good dropletplacement. A simple line pattern was prepared by SHOT miniconnected to the jetting machine with a controllable on-time,number of layers, and printing speed. Meanwhile, the off timeand air pressure was controlled by the air controller connectedto the jetting machine. The line pattern was selected during theoptimization of the printing setting.The printed line width and the gap were optimized bycalculating the results using Image-J soware. Off time wasvaried at 6, 7, 8, and 10 ms resulting in the line width (mm) of531 ± 23, 748 ± 19, 919 ± 40, and 965 ± 13, respectively, andline gap (mm) of 727 ± 50, 548 ± 86, 332 ± 49, and 319 ± 11,respectively. Those results showed that off-time affected thewidth size of the printed ice, which also affected the line gap.The lower-off time resulted in a fast-printing process witha smaller width of the ice line (higher resolution). Meanwhile,below 6 off time, the formed line was disconnected. Air pressure(MPa) was varied at 0.01, 0.008, 0.006, and 0.004 resulting in theline width (mm) of 1152 ± 41, 851 ± 76, 719 ± 48, and 697 ± 78,respectively, and line gap (mm) of 375 ± 25, 603 ± 32, 681 ± 47,and 736 ± 42, respectively. The air pressure affected the widthsize of the ice line, with higher air pressure resulting in a largerwidth of the ice line. This means that lower air pressure couldincrease the resolution of the printed ice, however, if the pres-sure is too low, no water droplets will be formed.© 2025 The Author(s). Published by the Royal Society of Chemistry3.2 Formation of scaffold with different surface patternsSHOT Mini, which was used to draw the pattern can be utilizedto form lines (X and Y direction), circles, and curves. Therefore,several patterns including big circles, small circles, horizontallines, and lattices could be prepared. By using printing condi-tions of 40 mm s−1 printing speed, 3 number of layers,0.006 MPa air pressure, and 8 ms off time, ultrapure water wasjetted onto a frozen copper plate wrapped with PFA lm andforming a different pattern of printed ice as shown in Fig. 2(upper row). The mixture of gelatin/ice particulates was addedonto the printed ice and then aer the lyophilization, thescaffold with different surface patterns was formed (Fig. 2(bottom row)).3.3 HUVEC viability and distribution on the scaffold withdifferent surface patternsUsing the optimum printing setting of 40 mm s−1 printingspeed, 3 layers, 0.006 MPa air pressure, and 8 ms off time, thedifferent pattern of big circle, small circle, line, and lattice wasobtained on the surface of scaffolds. The topographical struc-ture especially at the micro/nanoscale affects the behaviour ofHUVECs and plays an essential role in enhancing their perfor-mance on the biological function. HUVECs were seeded ontothe scaffold with different surface patterns and cultured for 1, 4,and 6 days. In comparison, the control gelatin scaffold withoutsurface pattern and ice particulates was prepared with the sameconditions of lyophilization, crosslinking, and sterilization.Live/dead staining was done to analyze the cells' viability aerdifferent culture times as shown in Fig. 3(A–C). The stainingshowed that most of the HUVECs were alive (green uores-cence) and few were dead (red uorescence) in the scaffold.These results showed that the viability of HUVECs cultured onthe scaffold was high. Aer 1 day of culture, the cell density onall samples was similar showing that different patterns did notaffect the initial attachment of HUVECs. However, aer theincrease of culture time, the cells' density on the big circle, line,and lattice pattern showed more than the small circle patternand control samples.Hoechst 33 528 staining was performed to analyze the cellnuclei of the HUVECs cultured on the scaffold aer 6 days asshown in Fig. 3(D). These nuclei staining showed the HUVECsalignment, which was affected by the surface pattern.RSC Adv., 2025, 15, 28581–28591 | 28585http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dFig. 3 Live/dead staining of HUVECs seeded on the scaffolds withdifferent surface pattern after (A) 1 day of culture, (B) 4 days of culture,(C) 6 days of culture, and (D) nucleus staining using Hoechst 33 528after 6 days of culture.RSC Advances PaperOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineCompared to the control ones, the porous scaffold prepared byice particulates showed homogeneous cell distribution. Morecells were spotted on the porous scaffold. The line and latticesurface of the scaffold, which had a smaller gap and sizecompared to the circle surface of the scaffold showed homo-geneous cell distribution.13F-actin staining was conducted to evaluate the elongationand spreading of HUVECs on different patterns of scaffolds'surface.12 Fig. 4 shows that HUVECs tend to elongate more andspread less on the patterned porous scaffolds compared to thecontrol scaffolds. It was because the porous construct disruptedthe surface geometry.24 Especially on the line pattern, cellelongation follows the direction of the line pattern. CircleFig. 4 F-actin staining using phalloidin of HUVECs seeded on thescaffolds with different surface patterns after 6 days of culture. The redsquare indicates the magnification area.28586 | RSC Adv., 2025, 15, 28581–28591patterns with a bigger size showed less effect on the cell'smorphology even though cell has receptive ability to a broadspectrum of size from macro to molecular level.25 The latticepatterns inuenced the volume fraction, which affected themechanical properties.26 Higher volume fraction on the latticepattern provided a larger cell area compared to the othersamples. Meanwhile, on the control scaffold, cells showedrandom orientation and maintained a large spreading. Thisresult has a similar performance to the previous study, whichinvestigated the HUVECs' behaviour on the patterned silkbroin lms.13 Their results discovered that the orientation andalignment of HUVECs are similarly impacted by graing ofdifferent diameters and the same depth. However, each type ofcell will react differently to topographic cues, and the shape,size, depths, and other characteristics should be controlled toguide the cells' behaviour. A similar result was reported on thealigned nanober samples, which showed a random orientationon un-patterned nanober.123.4 Formation of 3D composite sandwich scaffold withpatterned surface and interconnected porous structureAer freeze-drying, the 3D composite sandwich scaffold waspunched into a disc as shown in Fig. 5. A sandwich construct byputting the PLGA mesh at the center of a gelatin/nano-silicaporous scaffold was reported to have improved mechanicalproperties.19,27 The PLGA mesh serves as the structural frame-work of the scaffold, while the gelatin/nano-silica componentfacilitates cell accommodation.27 The interaction betweengelatin and nano-silica in composite scaffolds occurs primarilythrough hydrogen bonding between silanol groups (Si–OH) onthe nano-silica and amino or carboxyl group in the gelatinchains.28 As the matrix formulation, gelatin 4% with 70% iceparticulates was chosen as the optimum concentration, whichfacilitated the effective cell seeding and homogeneous ECMformation through scaffold based on the result from previouslyreported work by Shangwu Chen, et al.29 Their result showedthat high content of ice particulates ($70%) exhibited an openand well-interconnected pore network, which enabled uniformcell distribution. In contrast, scaffold prepared with lower iceparticulates ratio exhibited limited pore interconnectivity,Fig. 5 Appearance of the disc scaffold after water immersion (leftcolumn) and before water immersion (right column) during porositymeasurement. Scale bar = 1 mm.© 2025 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dFig. 6 SEM images of 3D composite sandwich scaffold and controlPaper RSC AdvancesOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlineresulting in limited cell penetration. Previous works hadexplored the use of these gelatin/ice particulates for differentbiomedical applications.30–33 As comparison, the control scaf-fold of gelatin/nano-silica/PLGA was prepared without surfacepattern and ice particulates. Both composite scaffold andcontrol scaffold in dry and hydrated states are shown in Fig. 5.The porosity of the composite and control scaffolds was calcu-lated using eqn (1) resulted in the porosity of 99.26 ± 1.00 mmand 97.19 ± 0.22 mm, respectively. The porous structure of thecomposite scaffold showed higher porosity compared to thecontrol scaffold. More water can be absorbed onto thecomposite scaffold, which is benecial for cell seeding and thetransfer of nutrients during culture.The stability of hydrated scaffold aer the water immersionis correlated with the covalent crosslink using EDC/NHS as thechemical crosslinker. EDC (1-ethyl-e-(3-dimethylaminopropyl)carbodiimide) activates carboxyl groups, enabling its conver-sion into an NHS-activated intermediate upon reaction withNHS, with urea as a by-product. This intermediate then reactswith a free amine group, resulting in the formation of a stableamide bond that serves as a crosslink between polymer chain.34scaffold at low magnification (upper row) and high magnification(lower row). The red dashed squares, yellow arrows, blue circles, andgreen circles indicate the magnification region, PLGA mesh, largepores, and interconnected pores, respectively.3.5 Morphology and pore sizeSEM analysis was performed to check the morphology andmicrostructures of the composite and control scaffolds asshown in Fig. 6. The surface images showed that the compositescaffold has open and interconnected pores with a line pattern.Meanwhile, the control scaffold has a skin layer and closedsurface pores. The calculation of pores size using image-J so-ware resulted in the line pattern of the composite scaffold'ssurface with a width of 534 ± 49 mm and a gap of 365 ± 28 mm.The pores were interconnected with a size of interconnectedpores of 34 ± 6 mm.The 3D-printed ice line could control the open pores on thesurface of the scaffold, which is benecial for the cells' pene-tration and distribution. The depth of the line well of thecomposite scaffold had the size of 146 ± 17 mm, which can becontrolled by adjusting the number of layers of 3D-printed ice.Besides the depth, the width and gap of the line pattern can beadjusted by setting the input on the Aerojet dispenser for 3D-printed ice formation. Previous works reported different sizesand gaps that can be achieved by this method and analyzed itsinuence on cells.15,16The horizontal and vertical cross-section images showed thatboth the composite and control scaffold had a spherical largepore. However, the composite scaffold showed a homogeneousstructure with interconnected pores. Meanwhile, the controlscaffold showed random pores with minimum interconnectedpores. The large pores of the composite scaffold were 164 ± 25mm and the interconnected pores were 36 ± 10 mm. The free iceparticulates could control the large pores size of the scaffolds,and the new ice crystals that were formed during the freezingprocess became the replica of the interconnected pores. Someprevious studies have explored the use of ice particulates asporogen materials to control the porous structure ofscaffolds.35–38 The pore size of scaffold affected compressive© 2025 The Author(s). Published by the Royal Society of Chemistrymodulus, which resulting in higher tissue formed in the scaf-fold.37 Previous research studied the effect of different iceparticulates size using four varied sizes, e.g. 150–250 mm, 250–355 mm, 355–425 mm, and 425–500 mm. Their results showedthat the highest compressive modulus of cartilage-like tissuewas obtained using 150–250 mm ice particulates. That can beexplained by the homogeneous cell distribution, where thesmaller pores facilitated more efficient cell inltrationcompared to the larger pores.39 In addition, another researchstudied the effects of gelatin:ice particulates ratio and showedthat the compression modulus of scaffold prepared by iceparticulates was more uniform and controllable than thecontrol scaffold prepared without the ice particulates. Thepresence of uniformly arranged and densely packed porescontributed to the mechanical reinforcement of the scaffold.38Meanwhile, the control scaffolds prepared without ice particu-lates exhibited a non-uniform porous architecture and aniso-tropic compressive modulus, indicating variability inmechanical performance depending on the direction ofcompression.29The vertical cross-section images also showed that PLGAknitted mesh was integrated well with the gelatin/nano-silicaboth on the composite and control scaffold. The integrationof PLGA as a supportive skeleton within the scaffold providessufficient mechanical strength to allow xation during suturingfor the clinical application. This is benecial to secure the graat the target site, which is still remains a signicant challenge,particularly when the scaffold materials is mechanically weak,such as on the gelatin-based construct.27 Our previous con-ducted research showed that PLGA improved the tensileRSC Adv., 2025, 15, 28581–28591 | 28587http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dRSC Advances PaperOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinestrength of the 3D composite sandwich scaffold.19 These nd-ings highlight the importance of both material compositionand pore structure in achieving mechanically stable scaffoldssuitable for load-bearing tissue regeneration.Fig. 8 (A) Live/dead staining, (B) nucleus staining by Hoechst 33 528and F-actin staining by phalloidin, (C) ALP staining, and (D) Alizarin redS staining after 7 day culture of hMSCs seeded on the 3D compositeand control scaffold.3.6 hMSCs attachment on the 3D composite sandwichscaffolds with patterned surface and interconnected porousstructureCells attachment and morphology aer 1 day of culture wereanalyzed using SEM as presented in Fig. 7. It showed the hMSCsattachment on both surfaces of the scaffolds. The surface imagepresented that the cells were merged and formed a cellularlayer.40 Meanwhile, some of the cells were migrated inside thescaffolds as shown in the horizontal and vertical cross-sectionimages. The vertical cross-section on the centre of the scaffoldshowed that more cells were observed on the composite scaffoldcompared to the control scaffolds. The interconnected porousstructure and open pores contributed to cell penetration on thecomposite scaffold.3.7 hMSCs viability, ALP, and alizarin red S analysis on the3D composite scaffolds with patterned surface andinterconnected porous structureLive/dead staining was performed to check the cell viability onthe scaffolds aer 7 days of culture as shown in Fig. 8(A). Thesurface images showed that hMSC were alive and attached bothon the composite and control scaffolds. Meanwhile, the verticalcross-section showed that more cells were spotted inside thecentre of composite scaffold compared to control scaffold. Atthe control scaffold, most of the cells were attached to thesurface and fewer cells were spotted at the centre of controlscaffolds.Hoechst 33 528 staining was performed to stain the cellnuclei, and phalloidin staining was conducted to stain F-actinof hMSCs seeded on the scaffold aer 7 days of culture(Fig. 8(B)). The surface images showed that cells were distrib-uted both on the surface of composite and control scaffolds.There was more void in the centre of control scaffolds withoutany cells. Besides the homogeneous cell distribution due to theinterconnected porous geometry on the composite scaffolds,the addition of nano-silica encouraged cell proliferation andFig. 7 SEM images of hMSCs seeded on the 3D composite and controlscaffold after 1 day of culture.28588 | RSC Adv., 2025, 15, 28581–28591improved the bioactivity and cellular behaviour.41 Those lead tostrong alkaline phosphatase and alizarin red S staining on thecomposite scaffolds as shown in Fig. 8(C and D), which indi-cates the osteogenic potential. The stronger ALP and Alizarinred S staining was observed on the composite scaffold,compared to the control scaffold. ALP provides an importantfunction to initiate matrix mineralization, which is benecialfor bone tissue regeneration.42 Meanwhile, the alizarin red Srevealed the existence of a hydroxyapatite layer and evaluatedthe level of mineralization.43 The dark red spots on the stainingshowed calcium deposition, which indicates enhanced miner-alization inside the scaffolds, which is stimulated by bioactivenano-silica.44Besides mineralization, vascularization plays a critical rolein bone tissue regeneration, which can be assessed by theangionenic promotion. Although direct angiogenic assays werenot included on this manuscripts, the early assessment of cellsproliferation of HUVECs on the patterned scaffold showedenhanced cell adhesion compared to the un-pattered controlscaffold. In addition, the previous studies have demonstratedthat the nano-silica inclusion on the gelatin scaffold enhancesangiogenic responses, including upregulated VEGF expressionand improved endothelial network formation.454 Conclusions3D composite gelatin/nano-silica/PLGA scaffold with surfacepatterning and interconnected porous architecture wassuccessfully fabricated by nozzle-based Aerojet dispenser 3Dprinting and freeze-drying method. Exploration of the printingprocess of water onto frozen substrate was performed to obtainhigh-resolution and stable printed ice as fugitive ink to forma patterned surface. Different patterns including the big circle,small circle, line, and lattice pattern of porous gelatin scaffoldwere fabricated using the optimum printing condition. In vitroculture of HUVECs onto a surface patterned scaffold was con-ducted to analyze the cells' behaviour and orientation. The© 2025 The Author(s). Published by the Royal Society of Chemistryhttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891dPaper RSC AdvancesOpen Access Article. Published on 12 August 2025. Downloaded on 9/9/2025 2:00:00 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlineaddition of printed ice and ice particulates could forma homogeneous interconnected pores structure in the matrixand controlled orientation surface pores. hMSCs were seededon the 3D composite scaffold and control scaffold prepared byfreeze-drying method. The cell attachment, cell viability, andcell distribution on the 3D composite scaffold were higher thanthe control scaffold due to its porous architecture. The additionof nano-silica showed an improved ALP and Alizarin red Sactivity, which initiated the matrix mineralization for bonetissue regeneration. Based on the early assessment on thisresearch using in vitro analysis on the bone regeneration usinghMSC and HUVECS, further in vivo analysis needs to be done inthe future work to support the study of angiogenesis andosteogenesis in bone tissue formation using this 3D compositescaffold.Author contributionsNur R. E. Putri designed and conducted the main experimentalwork on scaffold fabrication and characterization; HuajianChen carried out the experiments on the cell's characterization;Naoki Kawazoe contributed on the experimental process anddata analysis; Felicity R. A. J. Rose contributed on the supervi-sion; Ricky. D. Wildman contributed to the supervision;Guoping Chen contributed on the supervision and providingthe funding. All authors discussed the results and contributedto the writing, reviewing, and revising the manuscript.Conflicts of interestThe authors declare no conict of interest.Data availabilityThe supporting data for this study are available in themanuscript.AcknowledgementsThis research was partially supported by JSPS KAKENHI GrantNumber 24K03289.References1 L. Claes, S. Recknagel and A. Ignatius, Fracture healingunder healthy and inammatory conditions, Nat. Rev.Rheumatol., 2012, 8(3), 133–143, DOI: 10.1038/nrrheum.2012.1.2 J. A. Cottrell, J. C. Turner, T. L. Arinzeh and J. P. O. Connor,The Biology of Bone and Ligament Healing, Foot Ankle Clin.,2016, 21, 739–761, DOI: 10.1016/j.fcl.2016.07.017.3 B. Beamer, C. Hettrich and J. Lane, Vascular EndothelialGrowth Factor: An Essential Component of Angiogenesisand Fracture Healing, HSS J., 2015, 6(June), 85–94, DOI:10.1007/s11420-009-9129-4.4 M. Orciani, M. Fini, R. Di Primio and M. 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B, 2016,4(3), 505–512, DOI: 10.1039/c5tb02401c.RSC Adv., 2025, 15, 28581–28591 | 28591https://doi.org/10.1002/jcb.28768https://doi.org/10.1016/j.nano.2011.11.003https://doi.org/10.1039/c5tb02401chttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d5ra02891d Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering Tailoring cell behaviour by surface micropatterning and interconnected porous structure of gelatin/nano-silica/PLGA 3D composite scaffold for bone tissue engineering