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Bowu Peng, Huajian Chen, Chengyu Lu, [Tianjiao Zeng](https://orcid.org/0000-0002-1286-0337), Man Wang, [Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), Yingnan Yang, [Guoping Chen](https://orcid.org/0000-0001-6753-3678)

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[A Heparin‐Functionalized Scaffold with HB‐EGF Immobilization for Tissue Engineering](https://mdr.nims.go.jp/datasets/aa2c825b-af8b-42b3-9883-b1dafa1c982e)

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A Heparin‐Functionalized Scaffold with HB‐EGF Immobilization for Tissue EngineeringRESEARCH ARTICLEwww.advhealthmat.deA Heparin-Functionalized Scaffold with HB-EGFImmobilization for Tissue EngineeringBowu Peng, Huajian Chen, Chengyu Lu, Tianjiao Zeng, Man Wang, Toru Yoshitomi,Naoki Kawazoe, Yingnan Yang, and Guoping Chen*The introduction of growth factors (GFs) into scaffolds to mimic the in vivomicroenvironment is a promising approach for tissue engineering. In thisstudy, a heparin-functionalized scaffold is designed to mimic the GFs reservoirfunction of extracellular matrix (ECM). Owing to its heparin-binding domain,heparin-binding epidermal growth factor-like growth factor (HB-EGF) iseffectively and spatially captured by heparin-functionalized scaffold.Furthermore, the strong interaction between heparin and heparin-bindingdomain confers excellent stability of the immobilized HB-EGF in scaffold overa long period. The heparin-functionalized scaffold immobilized with HB-EGFfacilitates cell adhesion and promotes proliferation of human mesenchymalstem cells (hMSCs), while not inducing their differentiation duringproliferation. These results indicate that the immobilized HB-EGF has apromotive effect on proliferation of hMSCs without triggering spontaneousdifferentiation, and the system shows as a promising strategy to enhancestem cells proliferation in scaffolds.1. IntroductionTissue engineering (TE) has attracted considerable attention asa promising strategy for regeneration of functional tissues andorgans to treat human diseases and defects.[1–4] As a key ele-ment of TE, scaffolds provide structural support for cell adhe-sion and proliferation,[5] while offering a 3D space that mim-ics the in vivo extracellular microenvironment. Scaffolds shouldpossess porous and interconnected architectures to facilitate cellB. Peng, H. Chen, C. Lu, T. Zeng, M. Wang, T. Yoshitomi, N. Kawazoe,G. ChenResearch Center for Macromolecules and BiomaterialsNational Institute for Materials ScienceIbaraki 305-0044, JapanE-mail: Guoping.CHEN@nims.go.jpB. Peng, C. Lu, T. Zeng,M.Wang,G.ChenGraduate School of Science andTechnologyUniversity of TsukubaIbaraki 305–8577, JapanY. YangGraduate School of Life andEnvironmental ScienceUniversity of TsukubaIbaraki 305–8572, JapanThe ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/adhm.202502771DOI: 10.1002/adhm.202502771distribution, migration, adhesion, and pro-liferation, which can even determine cellu-lar fate during the process.[5–9]Mesenchymal stem cells (MSCs) arean attractive cell source for TE due totheir multipotent properties, includingself-renewal, plasticity, and multilin-eage differentiation.[10,11] MSC-based TErequires expansion of MSCs to reach suf-ficient numbers because patient-derivedMSCs are limited and decline with age,and their slow growth may impair thera-peutic efficacy.[10,12,13] MSCs cultured inporous scaffolds can proliferate to largenumbers and subsequentially differentiateinto functional tissues. During this pro-cess, promotion of cell proliferation in theporous scaffolds is critical. One effectivestrategy to enhance cell proliferation is theincorporation of bioactive compounds ormotifs into the porous structure.Bioactive factors (BFs) are molecules thatcan interact with tissues and regulate cell functions, includinggrowth factors (GFs), cytokines, and other biomolecules.[14,15]Among these, GFs have beenmost extensively studied in TE.[15,16]Despite their powerful therapeutic potential, their short half-lifeand low stability render the susceptibility of deactivation anddegradation in biological microenvironments (e.g., enzymatic ac-tivity), which hinder their further clinical application.[17,18] Theefficient delivery of GFs is essential for regulating cell func-tions, including migration, proliferation, survival, and differen-tiation, and thus plays a crucial role in tissue regeneration. Gen-erally, physical (e.g., adsorption or entrapment) and chemicalmethods (e.g., covalent binding) are commonly employed to loadGFs.[16,19,20] However, physical techniques often lead to burstand non-responsive release, while covalent bonding typically tar-gets in amino groups, which may alter the bioactivity of GFs orcause effects distinct from those of free GFs.[21] Potential over-dosing risks, non-responsive release, and bioactivity loss mayfurther induce therapeutic side effects.[16] Consequently, newplatforms are needed for efficient delivery of GFs. Bioaffinity-based methods and genetic engineering strategies are emergingas promising alternatives to sequester GFs and regulate cellularfunctions, thereby circumventing the drawbacks of conventionalmethods.[16,22–26]The extracellular matrix (ECM) is a 3D network surroundingcells in vivo and composed of diversemacromolecules. It not onlyparticipates in cellular processes but also serves as a reservoir forAdv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (1 of 11)http://www.advhealthmat.demailto:Guoping.CHEN@nims.go.jphttps://doi.org/10.1002/adhm.202502771http://crossmark.crossref.org/dialog/?doi=10.1002%2Fadhm.202502771&domain=pdf&date_stamp=2025-10-01www.advancedsciencenews.com www.advhealthmat.deFigure 1. Schematic illustration of the design and preparation of the composite system comprising a porous gelatin/heparin scaffold and HB-EGF fortissue engineering.GFs.[17] ECM contains abundant GFs such as insulin-like growthfactor, fibroblast growth factor, hepatocyte growth factor andtransforming growth factor-𝛽. These GFs associate with ECM tomaintain their activities and regulate cell functions such as rapidextracellular signaling activation, information transfer, functionmemory andmatrix homeostasis.[27] Moreover, the activity of GFsimmobilized by ECM can be controlled by this bioaffinity andresult in a longer and localized signaling activation.[27] As a re-sult, ECM-derived materials have been explored as ideal plat-forms for safe and economical delivery of GFs in TE.[28–30] Gly-cosaminoglycans (GAGs), along with their proteoglycan deriva-tives and glycoproteins, are key mediators of GFs sequestrationwithin the ECM.[20,31] Heparin, a linear polysaccharide andmem-ber of the GAGs family, possesses the highest negative chargedensity of any naturally derived biomolecules. It has been exten-sively applied in TE and drug delivery as an effective candidate forGFs loading. Heparin functions as an affinity-binding moleculeand has been shown to interact promiscuously with various GFs,thereby extending their half-lives and preserving bioactivity inbiological microenvironments.[32–36] Heparin-binding epidermalgrowth factor-like growth factor (HB-EGF), a member of the epi-dermal growth factor (EGF) family, regulates MSCs migration,proliferation, survival, and differentiation.[37–39] HB-EGF con-tains both an EGF-like domain and a heparin-binding (HB) do-main, conferring strong affinity for heparin or heparan sulfate.Gelatin, a hydrolyzed form of collagen, has also been widely usedfor scaffold preparation in 3D cell culture.[40–42]Therefore, in this study, a gelatin/heparin (GH) porous scaf-fold was designed and prepared by using pre-prepared ice par-ticles as a porogen reagent. HB-EGF was spatially sequesteredwithin the GH porous scaffold through affinity-binding betweenits HB domain and heparin. The efficiency and stability of the im-mobilizedHB-EGF in theGH scaffoldwere characterized, and itseffects on proliferation and differentiation of MSCs were system-atically evaluated.2. Results2.1. Preparation and Characterization of ScaffoldsThe composite scaffold was prepared by immobilizing HB-EGFin the GH porous scaffold. At first, GH scaffold was fabricatedby using ice particulates as a porogen reagent (Figure 1). Ice par-ticulates with diameters between 150 – 250 μm were adopted tocontrol the porous structure of the scaffold. The gelatin scaffold(G scaffold) without heparin was prepared as a control. The Gand GH scaffolds exhibited similar porous structures, with largespherical pores containing small pores on the large pore walls,indicating that all the scaffolds possessed well-interconnectedstructures. In addition, the addition of heparin did not influ-ence the microstructure of the scaffold (Figure 2a–d; Figure S1,Supporting Information). The size distribution of large sphericalpores in G and GH scaffolds was 191.6 ± 26.6 μm and 193.3 ±27.1 μm, respectively (Figure S2, Supporting Information). FT-IR (Figure S3, Supporting Information) showed the presenceof amide A peak at 3300 cm−1, representing the stretching fre-quency of N–H in amide groups. A strong amide II peak ≈1546 cm−1, attributed to the bending frequency of N–H conju-gated with C–O, was observed along with an amide I peak ≈1628 cm−1, indicating the stretching vibration of C─O in amidegroups. Peaks at 1105 and 983 cm−1 corresponded to the stretch-ing frequency of S = O and S–O in heparin.[43] In the GH scaf-fold, the correlative peaks of S═O and S─Owere observed≈ 1120and 973 cm−1, confirming the successful conjugation betweengelatin and heparin. The presence of heparin in the GH scaffoldwas further verified by Alcian blue staining.[44] The G scaffoldAdv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (2 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 2. Microstructures of the scaffolds: Scanning electron microscopy (SEM) images of horizontal cross-sections of a,b) G scaffold and c,d) GHscaffold; Alcian blue staining of e) G scaffold and f) GH scaffold.was not positively stained (Figure 2e), indicating the absence ofheparin in the gelatin control. In contrast, the GH scaffold waspositively stained (Figure 2f), and the homogeneity of staining in-dicated the homogeneous distribution of heparin throughout theGH scaffold (Figure 2f). Together, the Alcian blue staining andFT-IR results demonstrated hybridization of heparin and gelatin,confirming the successful preparation of GH scaffold.The mechanical properties of dry and hydrated scaffolds werealso measured (Figure S4, Supporting Information). The G andGH scaffolds had the same Young’s modulus under both dryand hydrated conditions.However, the hydrated scaffolds showedmuch lower Young’s modulus than the dry scaffolds. These re-sults indicated that the addition of heparin did not influence themechanical properties of porous scaffolds.2.2. Immobilization of HB-EGF in GH ScaffoldHeparin is a type of GAGs with a high negative charge dueto abundant carboxylic and sulfonate groups in its structure(Figure 3a).[45,46] HB-EGF, a member of the EGF family, containsa HB domain and an EGF-like domain (Figure 3b).[47,48] The HBdomain of HB-EGF plays critical role in its immobilization. Anal-ysis of the electrostatic potential ofHB-EGF indicated that theHBdomain carried a positive charge, enabling interaction with neg-atively charged heparin to form a stable complex through electro-static and hydrogen-bonding interactions (Figure 3b; Figure S5,Supporting Information).[49,50] Docking analysis of heparin andHB-EGF (Figure 3c) showed that the recognition of heparin byHB-EGF occurred specifically in the HB domain of the protein,where hydrogen bonds were formed. Localization of formed hy-drogen bonds between heparin and HB-EGF revealed the inter-actions were attributed to heparin-binding domain. These resultsdemonstrated the essential role of heparin-binding domain in es-tablishing a growth factor reservoir in this system.The successful immobilization of HB-EGF in the GH scaffoldwas examined by immunochemical staining of HB-EGF. The Gand GH scaffolds without HB-EGF were not positively stained(Figure 4a,d). The G scaffold incubated with HB-EGF solutionshowed slight staining, likely due to the physical absorption ofHB-EGF (Figure 4b). In contrast, the GH scaffold immobilizedwith HB-EGF exhibited very strong staining (Figure 4e), indicat-ing a large amount ofHB-EGFwas immobilized through the spe-cific interaction between heparin and HB-EGF. The long-termretention of immobilized HB-EGF in the GH scaffold was fur-ther investigated. After incubation in PBS for 7, 14, and 21 days,immunochemical staining of the GH scaffold immobilized withHB-EGF remained strong (Figure 4f; Figure S6, Supporting In-formation), demonstrating that the immobilized HB-EGF in GHscaffold was stable and could be retained for extended periods. Incomparison, the G scaffold coated with HB-EGF showed weak-ened staining after 7 days, indicating that the absorbed HB-EGFin the G scaffold was unstable and could be desorbed during in-cubation. Furthermore, ELISA analysis showed that the loadingefficiency of HB-EGF in the GH scaffold was 92.6 ± 0.1%, whichwas comparable to the previously reported results.[51] Collectively,these findings demonstrated that the GH scaffold could signifi-cantly enhance the loading and retention of HB-EGF.2.3. Cell Adhesion, Distribution, and Viability in ScaffoldsNext, hMSCs were seeded and cultured in the scaffolds. Thecell seeding efficiency in the GH (-) (GH) and GH (+) (GH-Adv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (3 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 3. Structure of heparin and HB-EGF: a) Chemical andmodeling structure of heparin; b) Modeling structure, electrostatic distribution and peptidesequence of HB-EGF and c) Interaction analysis of docking complex. Yellow dotted line: hydrogen bonds formed between heparin and HB-EGF.HB-EGF) scaffolds was 96.3 ± 1.6% and 95.9 ± 1.1%, respec-tively (Table S1, Supporting Information). All scaffolds showedhigh seeding efficiency due to their interconnected microp-orous structures. As shown in Figure 5a,d, cells adhered to thesurfaces of the interconnected pores in the GH and GH-HB-EGF scaffolds. The two scaffolds showed similar adhesion re-sults, and the immobilized HB-EGF did not affect adhesionproperties. After 7 days (Figure 5b,e) and 21 days (Figure 5c,f)of culture, more cells were observed in the pores of scaf-folds compared to 1 day of culture. After 21 days of culture,more cells were observed in the GH-HB-EGF scaffolds thanin the GH scaffolds, indicating cell proliferation in the scaf-folds.Cell distribution in the scaffolds was assessed by nucleus stain-ing (Figure 5g–l). The nuclei were homogeneously stained fromthe top surface to the bottom of all scaffolds, showing uniformcell distribution in all scaffolds after 1 day of culture (Figure 5g–i).After 3 days, the cells remained homogeneously distributed in allscaffolds (Figure 5j–l), indicating that the immobilized HB-EGFdid not affect cell distribution. The similar microporous struc-tures of all scaffolds likely explained this observation. The highcell seeding efficiency and homogenous distribution of cells wereascribed to the well-interconnected porous structures of the scaf-folds.Cell viability in the scaffolds was investigated by live/deadstaining (Figure 6a–f). Nearly all cells were alive after 1 day(Figure 6a–c) and 3 days (Figure 6d–f) of culture. Even after 7and 21 days, almost all cells remined viable (Figure S7, Sup-porting Information). The cell density increased after 21 days ofculture due to cell proliferation in the scaffolds. These resultsindicated that the cells maintained high viability and proliferatedin all scaffolds.Adv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (4 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 4. Immunochemical staining of the scaffolds. a) G scaffold without HB-EGF; b) G scaffold incubated with HB-EGF immediately after prepara-tion; c) G scaffold incubated with HB-EGF after 7 days; d) GH scaffold without HB-EGF; e) GH scaffold immobilized with HB-EGF immediately afterpreparation; f) GH scaffold immobilized with HB-EGF after 7 days. (-): without HB-EGF; (+): with HB-EGF.2.4. Effect of Immobilized HB-EGF on Cell Proliferation andDifferentiationQuantification of DNA amount (Figure 7a) was used to comparecell proliferation in the GH andGH-HB-EGF scaffolds. TheDNAamount in GH-HB-EGF scaffolds was significantly higher thanin GH scaffolds, suggesting that the GH-HB-EGF scaffolds en-hanced cell proliferation more effectively. The immobilized HB-EGF could promote hMSCs proliferation during 3D culture inthe porous scaffold. In addition, the effects of immobilized HB-EGF and free HB-EGF on proliferation enhancement of hMSCswere also evaluated (Figure S8, Supporting Information). Boththe free and immobilized HB-EGF groups showed higher DNAamount than the groupwithoutHB-EGF. Comparedwith the freeHB-EGF, the immobilized HB-EGF induced a more sustainedproliferation enhancement of hMSC, likely due to its prolongedhalf-life and preserved bioactivity conferred by heparin binding,which may extend EGFR activation and thereby promote greatercellular proliferation.[51–53]To evaluate the effects of immobilized HB-EGF on differen-tiation of hMSCs, chondrogenic differentiation was first inves-tigated. The sGAG amount of hMSCs after 21 days of culturewas measured (Figure 7b,c). The sGAG amount in both GH andGH-HB-EGF scaffolds increased over time. At each time point,the sGAG amount in the GH-HB-EGF scaffolds was signifi-cantly higher than in the GH scaffolds. However, the sGAG/DNAratio, representing the sGAG production per cell, did not dif-fer significantly between hMSCs cultured in the GH and GH-HB-EGF scaffolds (Figure 7c). These results indicated that hM-SCs in both scaffolds had the same capacity for sGAG produc-tion. Thus, immobilized HB-EGF did not affect sGAGs synthe-sis.Furthermore, expression of chondrogenic genes was analysedby RT-qPCR after hMSCs were cultured in the scaffolds for 3,7, 14, and 21 days (Figure 7d–g). Four chondrogenic relatedgenes, type I collagen (COL I), type II collagen (COL II), aggre-can (ACAN), and SRY-box transcription factor 9 (SOX 9), wereselected for analysis. Expression levels of COL I, COL II, ACAN,and SOX 9 showed no significant difference between GH andGH-HB-EGF scaffolds at any time point. Moreover, the expres-sion of COL II was very low. Collectively, these results indicatedthat the immobilized HB-EGF did not affect chondrogenic differ-entiation of hMSCs. Although the immobilized HB-EGF showedpromotive effect on the proliferation of hMSCs during 3D culturein the porous scaffold, it had no influence on the chondrogenicdifferentiation of hMSCs.And then, the effects of immobilized HB-EGF on osteogenicand adipogenic differentiation of hMSCs during 21 days of cul-ture were also evaluated (Figure S9, Supporting Information).The osteogenesis-related genes, alkaline phosphatase (ALP) andrunt-related transcription factor 2 (RUNX2), were used for eval-uation of osteogenic differentiation. The adipogenesis-relatedgenes, fatty acid binding protein-4 (FABP4) and lipoprotein li-pase (LPL), were used for evaluation of adipogenic differentia-tion. The expression level of these genes in hMSCs cultured inGH-HB-EGF scaffolds was very low. These results indicated thatthe GH-HB-EGF did not induce osteogenic or adipogenic differ-entiation of hMSCs. Taken together, the findings demonstratedAdv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (5 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 5. Cell adhesion and distribution in the scaffolds. a–f) SEM images of scaffolds after hMSCs were cultured for 1 day: a) GH (-) scaffold and d)GH (+) scaffold; 7 days: b) GH (-) scaffold and e) GH (+) scaffold; and 21 days: c) GH (-) scaffold and f) GH (+) scaffold. Arrows indicate the cells. g-l)Nucleus staining of hMSCs cultured in scaffolds for 1 day: g) G (-) scaffold, h) GH (-) scaffold and i) GH (+) scaffold; 3 days: j) G (-) scaffold, k) GH (-)scaffold and l) GH (+) scaffold. Blue fluorescence indicates cell nuclei. (-): without HB-EGF; (+): with HB-EGF.Figure 6. Cell viability in the scaffolds. Live/dead staining of hMSCs after 1 day of culture: a) G (-) scaffold, b) GH (-) scaffold and c) GH (+) scaffold; after3 days of culture: d) G (-) scaffold, e) GH (-) scaffold and f) GH (+) scaffold. Green fluorescence indicates living cells while red fluorescence indicatesdead cells. (-): without HB-EGF; (+): with HB-EGF.Adv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (6 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 7. Proliferation and chondrogenic differentiation of hMSCs in the scaffolds. a) DNA quantification; and b) sulfated glycosaminoglycan (sGAG)quantification; c) sGAG/DNA ratio; d–g) Gene expression levels of COL I, COL II, SOX 9 and ACAN in hMSCs cultured in GH (-) and GH (+) scaffoldsfor 3, 7, 14, and 21 days. Data are presented as the mean ± SD (n = 3). Significant differences: *p < 0.05; **p < 0.01; ***p < 0.001; ns., not significant.(-): without HB-EGF; (+): with HB-EGF.that the immobilized HB-EGF did not trigger chondrogenic, os-teogenic or adipogenic differentiation of hMSCs.2.5. In Vivo Efficacy and Biosafety Evaluation of ScaffoldsSubcutaneous implantation was performed to investigate the invivo efficacy and biosafety of the scaffolds. The hMSCs wereseeded in the GH scaffolds and GH-HB-EGF scaffolds and cul-tured for 3 days. The scaffolds were then subcutaneously im-planted in the backs of nude mice for 10 days. The GH andGH-HB-EGF scaffolds were harvested and examined by live/deadstaining and HE staining. The scaffolds shrank after 10 daysof implantation (Figure 8a,b), likely due to suppression by sur-rounding tissues, as hydrated scaffolds had weak mechanicalproperties. Live/dead staining indicated that the cells in GH andGH-HB-EGF scaffolds remained viable (Figure 8c). HE stainingshowed that more cells were detected in the GH-HB-EGF scaf-folds than in the GH scaffolds (Figure 8d). Furthermore, the ma-jor organs including heart, liver, spleen, lung and kidney fromthe mice implanted with GH-HB-EGF scaffolds, as well as frommice without implantation, were cross-sectioned and examinedby HE staining. Gross appearance and histology showed no sig-nificant differences between the organs from themice implantedwith GH-HB-EGF scaffolds and the mice without implantation(Figure 8e; Figure S10, Supporting Information). These resultsdemonstrated the in vivo efficacy and biological safety of the GH-HB-EGF scaffolds.3. DiscussionsIn this study, the GH porous scaffold was prepared for immobi-lization of HB-EGF to generate GH-HB-EGF composite scaffold.Heparin in the GH scaffold specifically bound to the HB-EGF,resulting in a high loading efficiency of HB-EGF in the GH scaf-fold. The immobilized HB-EGF was retained in the scaffold fora long period due to the stable bioaffinity binding between hep-arin and HB-EGF. More importantly, the extended half-life andpreserved bioactivity of immobilized HB-EGF, provided throughheparin interaction, allowed more efficient and economical useof HB-EGF in biomedical applications.The immobilized HB-EGF promoted the proliferation of hM-SCs but did not induce their differentiation. These findings wereconsistent with the previous results of free HB-EGF on the func-tions of MSCs.[37,39] Previous studies have reported that HB-EGFbinds to EGFR (HER1), which subsequently result in phosphory-lation of tyrosine residues in the receptor kinase domain, therebyactivating the downstream Akt and Erk1/2 pathways to regulateAdv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (7 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deFigure 8. In vivo efficacy and biosafety of scaffolds. a) Gross appearance of scaffolds before implantation; b) Gross appearance of implants after 10 daysimplantation; c) Live/dead staining of implants; d) HE staining of implants; e) HE staining of heart, liver, spleen, lung and kidney from the mice withoutor with cell/scaffold implantation for 10 days. (-): without HB-EGF; (+): with HB-EGF.cells proliferation.[48,54,55] Additionally, HB-EGF has been shownto inhibit spontaneous chondrogenic, osteogenic and adipogenicdifferentiation of MSCs.[37,39,56] This inhibitory effect on differ-entiation is temporary and reversible, depending on the pres-ence and dosage of HB-EGF.[37] The preliminary treatment withHB-EGF is necessary to hamper the differentiation cascade andthe block effect disappears after removal of HB-EGF.[37] Dur-ing this process, HB-EGF-mediated suppression of MSCs dif-ferentiation likely occurs through inhibition of BMP-Smad1/5/8signalling,[39] and the BMP-Smad1 pathway plays a critical role inosteoblast and chondrocyte differentiation.[57–60] The efficacy andbiosafety of this system were also explored in vivo, and the re-sults demonstrated the potential of this design for future clinicalapplication.In this study, we only investigated the effects of immobilizedHB-EGF on human bone marrow-derived MSCs. The effectsof immobilized HB-EGF on other types of stem cells, such asadipose-derived MSCs, require further investigation. Neverthe-less, immobilization of HB-EGF in GH scaffold proved to be auseful method for maintaining the bioactivity of growth factorsand promoting proliferation of stem cells during 3D culture inporous scaffolds, which is a critical process for TE. Furthermore,there are other BFs that, similar to HB-EGF, interact with hep-arin, and their bioactivities may also be enhanced by formingheparin-BF complexes.[61,62] The system developed in this studymay therefore offer broader and multifunctional applications inTE.4. ConclusionGH scaffolds were prepared by an ice particulates method tomimic the growth factor reservoir function of the ECM. Theporous structure was controlled by ice particulates, and the scaf-fold featured large spherical pores with good interconnectivity.The addition of heparin significantly enhanced the binding ca-pacity and stability of HB-EGF in the scaffold, resulting in theformation of GH-HB-EGF scaffold. The prepared GH-HB-EGFscaffold supported efficient cell seeding, homogenous cell dis-tribution, and enhanced proliferation of hMSCs, while not in-ducing their differentiation. Therefore, this system is expectedto serve as a base platform for introducing bioactive factors intoporous scaffolds for TE applications.5. Experimental SectionMaterials: Gelatin (type B from bovine skin) and heparin-binding epi-dermal growth factor-like growth factor (HB-EGF) were purchased fromSigma–Aldrich. Heparin sodium, 1-ethyl-3-(3-dimethylaminopropyl) car-bodiimide (EDC), N-hydroxysuccinimide (NHS), acetic acid and ethanolwere obtained from Wako Pure Industries, Ltd. All reagents were used di-rectly unless otherwise indicated.Preparation of Gelatin/Heparin (GH) and Gelatin (G) Scaffolds: TheGH porous scaffold was prepared by an ice particulates-based method ac-cording to the previous work[6,63,64] with some changes. Briefly, ice partic-ulates were acquired by spraying pure water into liquid nitrogen and the iceparticulates with diameters of 150–250 μm were sieved in a low tempera-Adv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (8 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dewww.advancedsciencenews.com www.advhealthmat.deture chamber. Then, the gelatin and heparin sodiumwere dissolved in 35%(v/v) acetic acid solution, respectively and the two solutions were mixedto form the homogeneous solution which contained 8% (m/v) gelatin and0.4% (m/v) heparin sodium. Subsequently, the temperature of themixturesolution and ice particulates were balanced at −4 °C for 6 h. And then, theice particulates were added into the mixture solution at a ratio of 7 (ice):3(solution) (m/v) and the final mixture was transferred into a silicon mold.The construct was placed in a −80 °C freezer for 12 h for freezing. Fi-nally, the frozen construct was lyophilized and crosslinked with a series ofethanol solutions containing EDC and NHS. After cross-linking, the scaf-fold was washed with MilliQ water to obtain the GH composite scaffold. Acontrol G scaffold without heparin was also prepared by the samemethod.Microstructure Characterization and Heparin Staining: The microstruc-tures of scaffolds were evaluated by scanning electron microscopy (SEM,JSM-IT800, Tokyo, Japan). The pore sizes of the scaffolds were mea-sured by using an ImageJ software to calculate the average diametersof 100 pores from each of three SEM images. The heparin in the scaf-folds was stained according to a previously reported method.[44] Briefly,the gelatin/heparin composite scaffold and gelatin control scaffold weretreated with Alcian Blue 8GX (0.5% w/v) in 3% acetic acid for 30 min andwashed by MilliQ water for three times. And then, the stained sampleswere lyophilized and characterized by an optical microscope.Characterization of Mechanical Property of Scaffolds: The mechanicalproperty of scaffolds was evaluated by Young’s modulus of scaffolds. Thescaffolds were punched into cylinder with a diameter of 6 mm and thick-ness of 6mm. The Young’smodulus of the scaffolds wasmeasured in bothdry and hydrated status. The hydrated scaffolds were prepared by immers-ing the scaffolds into PBS for 2 h at room temperature. The samples werecompressed at 0.1 mm s−1 by texture analyzer (TA, XTPlus. Texture Tech-nologies, Hamilton, MA, USA). The initial linear region of the stress-straincurves was chosen to calculate the Young’s modulus, and three samplesfrom each group were investigated for the evaluation.HB-EGF Immobilization and Evaluation—HB-EGF Immobilization:The G and GH scaffolds were punched into cylinder discs (𝜑 6 × 3 mm),and the discs were sterilized with 70% (v/v) ethanol aqueous solution for30 min. After washing with sterilized MilliQ water for 3 times, the scaffolddiscs were immersed in HB-EGF solution (100 ng mL−1) under shakingat 4 °C for 24 h. And then, the treated scaffolds were washed by sterilizedPBS for 3 times and stored at −20 °C for further use.HB-EGF Immobilization and Evaluation—Immunochemical Staining ofHB-EGF: Firstly, the GH scaffolds immobilized with HB-EGF were incu-bated in PBS containing 2% bovine serum albumin 4 °C for overnight fornon-specific antigen blocking. After that, the scaffolds were incubated withanti-HB-EGF primary antibody (Human HB-EGF Antibody, AF-259-NA,biotechne R&D SYSTEM) at 4 °C for overnight. After three PBS washes,samples were incubated with the Donkey Anti-Goat IgGH&L at room tem-perature for 2 h, followed by three further PBS washes. Color developmentwas performed with 3, 3- diaminobenzidine (DAB) for 5 min, and sampleswere characterized by an optical microscopy. The control G scaffold thatwas incubated with HB-EGF (100 ng mL−1, 4°C, 24 h) and washed withPBS was used to assess HB-EGF adsorption.HB-EGF Immobilization and Evaluation—Loading Efficiency of HB-EGF:Residual HB-EGF solution and PBS eluates from immobilization andwashing were collected and HB-EGF concentration in the collected solu-tion was measured by an ELISA test (Human HB-EGF Quantikine ELISAKit, biotechne R&D SYSTEM). The immobilized amount of HB-EGF in thescaffolds was calculated by subtracting the residual HB-EGF amount fromthe initial input. The loading efficiency was calculated by dividing the im-mobilized amount of HB-EGF in the scaffolds with input amount. Quadru-plicate samples were analyzed to calculate the mean values and standarddeviations.HB-EGF Immobilization and Evaluation—Stability of Immobilized HB-EGF: The G and GH scaffolds incubated with HB-EGF were placed inPBS under shaking (60 rpm) at 37 °C for 7 days to assess the stabilityof immobilized HB-EGF. The PBS was replaced with fresh solution every3 days. After 7 days, scaffolds were collected and evaluated by immuno-chemical staining ofHB-EGF. To evaluate longer term stability, GH scaffoldsamples were also examined after 14 and 21 days.Computational Modeling of Heparin and HB-EGF—Modeling of Heparin:The heparin model was generated by GLYCAM Web Tools (http://glycam.org).Computational Modeling of Heparin and HB-EGF—Modeling of HB-EGF:Modeling ofHB-EGFwas based on the peptide sequences ofHB-EGF[47,49]and constructed with SWISS-MODEL.[65–69] Electrostatic potential distri-bution of HB-EGF was calculated by PyMOL software (The PyMOLMolec-ular Graphics System, Version 2.6 Schrödinger, LLC).Computational Modeling of Heparin and HB-EGF—Molecular Dockingof Heparin and HB-EGF: Pre-processing of docking was based on anAutoDockTools[70] and binding of heparin to HB-EGF was analyzed by anAutoDock.[71,72,70,73] The semi-flexible docking was used in the dockingprocess. Heparin was treated as a completely flexible part and HB-EGF asa rigid part.[50] The coordinates of the central grid point of maps were de-fined as follows: x = −30.277, y = −8.034, z = 38.953 and the grid spacingwas 0.375 Å. The Lamarckian genetic algorithm with an initial populationsize of 300 and a termination condition of 27 000 generations and 2.5 ×107 energy evaluations were used. A total of 50 independent runs werecarried out.Cell Culture and Functional Evaluations—Culture of hMSCs in Scaffolds:The scaffold discs (𝜑 6 × 3 mm) were sterilized with a 70% (v/v) ethanolaqueous solution for 30 min and rinsed with sterilized MilliQ water for3 times. Human bone-marrow-derived mesenchymal stem cells (hMSCs)at passage 2 obtained from Lonza (Walkersville MD, USA) were subcul-tured in MSCGM medium. The hMSCs after two passages were collectedby trypsin treatment and resuspended in culture medium at a cell con-centration of 5 × 106 cells mL −1. A total of 84 μL cell suspensions wereadded onto the top side of the scaffold discs and cultured for 6 h. After 6h culture, the scaffold discs were turned upside down and the other sidewas also seeded with another 84 μL cell suspensions. After an additional 6h, samples were transferred to flasks containing DMEM (2.5% FBS, 4mmL-Glutamine, and 100 UmL−1 Penicillin-Streptomycin) for continuing cul-ture under shaking at 60 rpm. During cell seeding, the unadheredMSCs inthe seedingmediumwere collected and counted for calculation of seedingefficiency. For cells cultured in GH scaffold with immobilized HB-EGF, theconcentration of HB-EGF in the culture system was 25 ng mL−1.Cell Culture and Functional Evaluations—Evaluations of hMSCs Adhe-sion, Viability, Distribution and Proliferation: The hMSCs cultured in scaf-folds for 1, 7, and 21 days were observed by SEM. Briefly, the scaffoldswere washed with PBS and fixed with 2.5% glutaraldehyde for 1h. Af-ter fixation, the scaffolds were washed with MilliQ water and lyophilized.The lyophilized scaffolds were coated with platinum for SEM observation.Live/dead staining was conducted for evaluating cell viability and nucleistaining was conducted to examine cell distribution in the scaffolds. Afterculture for 1 and 3 days, the cells were stained by using a Live/dead cellstaining kit and Hoechst 33258. The stained samples were observed by afluorescence microscope.Cell Culture and Functional Evaluations—Evaluation of Immobilized HB-EGF and Free HB-EGF on hMSCs Proliferation: The hMSCs were seededin G and GH-HB-EGF scaffolds at equal densities. The cell-G scaffoldswere divided into two groups: G scaffold without HB-EGF and G scaffoldsupplemented with free HB-EGF. The concentration of HB-EGF in free andimmobilized groups was designed at 25 ng mL−1. After 7 days of culture,three groups were collected, and DNA amount was measured. Triplicatesamples were used for each measurement.Cell Culture and Functional Evaluations—Quantification of DNA and Sul-fated Glycosaminoglycan (sGAG): Proliferation of hMSCs in scaffolds wasevaluated by quantifying the DNA amount. GH scaffolds without HB-EGFwere used as a control. The cell/scaffold constructs after cell seeding andculture for 3, 7, 14 and 21 days were harvested for quantification of DNAand sGAG amounts. The harvested samples were washed, freeze-driedand digested with papain solution at 60 °C under shaking for 12 h. The pa-pain solution was prepared by dissolving papain in 0.1M PBS buffer (400μgmL−1) with L-cysteine hydrochloridemonohydrate (5mm) and EDTA (5mm) at a pH of 6. The amount of DNA and sGAG in the digestion solutionwas quantified with Hoechst 33258 and a BlyscanTM GlycosaminoglycanAssay Kit, respectively. Triplicate samples were analyzed to calculate themean values and standard deviations.Adv. Healthcare Mater. 2025, e02771 © 2025 Wiley-VCH GmbHe02771 (9 of 11) 21922659, 0, Downloaded from https://advanced.onlinelibrary.wiley.com/doi/10.1002/adhm.202502771 by Guoping Chen - National Institute For , Wiley Online Library on [13/10/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.advhealthmat.dehttp://glycam.orghttp://glycam.orgwww.advancedsciencenews.com www.advhealthmat.deReal-Time PCR: Samples cultured for 3, 7, 14, and 21 days werecollected for analysis of gene expression. The hMSCs cultured in theGH scaffolds without HB-EGF were used as a control. Samples werewashed three times with PBS and frozen in liquid nitrogen. The frozensamples were pulverized with a crusher and lysed with a Sepasol-RNAI Super G solution (1 mL). Total RNA was extracted according to areported protocol.[74] After reverse transcription with a high-capacitycDNA reverse transcription kit, amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene), collagentype I (Col1a2), collagen type II (Col2a1), aggrecan (ACAN) andSOX9 was conducted with a QuantStudio 3 Real-Time PCR System(Thermo Fisher Scientific). The previously reported primer and probesequences as shown below were used.[75,76] GAPDH: (forward) 5′-ATGGGGAAGGTGAAGGTCG-3′, (reverse) 5′-TAAAAGCAGCCCTGGTG-ACC-3′, (probe) 5′-CGCCCAATACGACCAAATCCGTTGAC-3′; Col1a2:(forward) 5′-CAGCCGCTTCACCTACAGC-3′, (reverse): 5′-TTTTGTATT-CAATCACTGTCTTGCC-3′, (probe): 5′−CCGGTGTGACTCGTGCAGCCATC-3′; Col2a1: (forward) 5′GGCAATAGCAGGTTCACGTACA-3′, (reverse)5′-CGATAACAGTCTTGCCCCACTT-3′, (probe) 5′−CCGGTATGTTTCGTG-CAGCCATCCT-3′; ACAN: (forward) 5′-TCGAGGACAGCGAGGCC-3′,(reverse) 5′-TCGAGGGTGTAGCGTGTAGAGA-3′, (probe) 5′-ATGGAAC-ACGATGCCTTTCACCACGA-3′; SOX9: (forward) 5′-CACACAGCTCACTCG-ACCTTG-3′, (reverse) 5′-TTCGGTTATTTTTAGGATCATCTCG-3′, (probe)5′-CCCACGAAGGGCGACGATGG-3′. For adipogenic differentiation,primer and probe sequences were used: FABP4 (Hs00609791_m1, Lot:1909263, Applied Biosystems), and LPL (Hs00173425_m1, Lot: 1953315,Applied Biosystems); For osteogenic differentiation, primer and probesequences were used: ALPL (Hs01029144_m1, Lot: 2103375, AppliedBiosystems) and RUNX2 (Hs00231692_m1, Lot: 2080647, AppliedBiosystems). A 2 −∆∆Ct method was used to calculate the relative expres-sion of each gene with endogenous control (GAPDH). Expression levelswere normalized against those of control group. Triplicate samples wereanalyzed to calculate the mean values and standard deviations.Subcutaneous Implantation: All in vivo experiments were conductedwith the approval from the Ethical Committee of Animal Experiments ofNIMS (accreditation No: 76-2023-12) and according to the CommitteeGuidelines. hMSCs seeded scaffolds cylinders (𝜑 6 × 3 mm) of GH andGH-HB-EGF were cultivated in vitro for 3 days before implantation. Nudemice obtained from Charles River Laboratories (Yokohama, Japan) wereused for subcutaneous implantation. The mice were sacrificed to collectthe samples and organ tissues after 10 days (n= 3). The samples collectedwere washed with PBS 3 times and fixed with 10% neutral buffered forma-lin for 24 h at room temperature. The fixed samples were dehydrated, em-bedded in paraffin and sliced with microtome to obtain slice with a thick-ness of 5 μm. The slices were stained with HE and evaluated by opticalmicroscopy.Statistical Analysis: The quantitative data were statistically analyzed byone-way analysis of variance (ANOVA) with Tukey’s post hoc test usingGraphPad Prism software (GraphPad Software, Boston, MassachusettsUSA, www.graphpad.com). The data are shown as the mean ± standarddeviations (S.D.) (n = 3) and significant differences are expressed as *(p < 0.05), ** (p < 0.01), and *** (p < 0.001).Supporting InformationSupporting Information is available from the Wiley Online Library or fromthe author.AcknowledgementsThis work was supported by JSPS KAKENHI Grant Number 24K03289 andJST SPRING Grant Number JPMJSP2124.Conflict of InterestThe authors declare no conflict of interest.Data Availability StatementThe data that support the findings of this study are available from the cor-responding author upon reasonable request.KeywordsHB-EGF, heparin, hMSCs, immobilization, porous scaffold, tissue engi-neeringReceived: June 2, 2025Revised: September 19, 2025Published online:[1] A. Atala, Tissue Eng., Part A 2024, 30, 5.[2] M. Risbud, Biogerontology 2001, 2, 117.[3] J. F. Mano, R. L. Reis, J. Tissue. Eng. Regen. Med. 2007, 1, 261.[4] C. W. Patrick Jr, Anat. Rec. 2001, 263, 361.[5] Q. L. Loh, C. Choong, Tissue Eng., Part B 2013, 19, 485.[6] Q. Zhang, H. Lu, N. Kawazoe, G. 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