Restricted accessResearch articleFirst published online 2025-8
Analysis of foreign body response and systemic toxicity of additively manufactured nanocellulose reinforced alginate gelatin-based scaffolds with interconnected 3D porous structure
The last two decaes have witnessed significant efforts to develop gelatin/alginate based scaffolds using variants of 3D printing techniques. However, their biocompatibility for regenerating complex soft tissues remains insufficiently explored. Addressing this gap, we fabricated 3D-printed alginate-gelatin (3A5G) and nanocellulose-reinforced (3A5G1C) hydrogel scaffolds with clinically relevant dimensions (15 mm diameter, 5 mm height) and the host tissue responses were critically analyzed. The distinct advantages of nanocellulose in modulating mechanical strength, viscoelasticity, swelling, and degradation characteristics were established in our prior studies. This investigation aimed to comprehensively evaluate the foreign body response of these scaffolds in a rat model. The animals exhibited healthy metabolic activity, evidenced by progressive weight gain, localized tissue healing, and normal mobility over 30 days. Histological analyses could not reveal any adverse immune reaction at 7- or 30-days, post-implantation. Hematological and serum biochemical assessments indicated a progression from acute (7 days) to sub-acute (30 days) inflammation, following subcutaneous implantation, without any signature of systemic toxicity. Immune marker evaluation (TNF-α, CD-8, CD-68, COX-2, IL-6) confirmed the absence of pathological immune responses, even with nanocellulose incorporation. Immunohistochemical analysis using CD31 staining demonstrated enhanced vascularization in nanocellulose-reinforced scaffolds at both 7 and 30 days. The absence of systemic toxicity from scaffold degradation products and the favorable biocompatibility outcomes underline the potential of these hydrogel scaffolds for soft tissue regeneration. The incorporation of nanocellulose further enhanced the scaffolds’ functional performance, particularly in promoting vascularization, positioning them as promising candidates for complex tissue engineering applications.
BasuB. Biomaterials science and tissue engineering: principles and methods. Cambridge, UK: Cambridge University Press, 2017.
2.
BasuB. Biomaterials science and implants. Berlin, Germany: Springer, 2020.
3.
BasuBGhoshS. Biomaterials for musculoskeletal regeneration. Berlin, Germany: Springer, 2017.
4.
ChowdhurySRKeshavanNBasuB. Urinary bladder and urethral tissue engineering, and 3D bioprinting approaches for urological reconstruction. J Mater Res2021; 36(19): 3781–3820.
5.
ChowdhuryRoySMondalG, et al.Three-dimensional extrusion printed urinary specific grafts: mechanistic insights into buildability and biophysical properties. ACS Biomater Sci Eng2024; 10(2): 1040–1061.
6.
DasSJegadeesanJTBasuB. Gelatin methacryloyl (GelMA)-based biomaterial inks: process science for 3D/4D printing and current status. Biomacromolecules2024; 25(4): 2156–2221.
7.
YuanXZhuWYangZ, et al.Recent advances in 3D printing of smart scaffolds for bone tissue engineering and regeneration. Adv Mater2024; 36: 2403641.
8.
HasanzadehRMihankhahPAzdastT, et al.Biocompatible tissue-engineered scaffold polymers for 3D printing and its application for 4D printing. Chem Eng J2023; 476: 146616.
9.
VacantiJPLangerR. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet1999; 354: S32–S34.
10.
O’BrienFJ. Biomaterials & scaffolds for tissue engineering. Mater Today2011; 14(3): 88–95.
11.
WangWDaiJHuangY, et al.Extracellular matrix mimicking dynamic interpenetrating network hydrogel for skin tissue engineering. Chem Eng J2023; 457: 141362.
12.
EltomAZhongGMuhammadA. Scaffold techniques and designs in tissue engineering functions and purposes: a review. Adv Mater Sci Eng2019; 2019(1): 1–13.
13.
MehrabiAZare JaliseSAhmadH, et al.Evaluation of inherent properties of the carboxymethyl cellulose (CMC) for potential application in tissue engineering focusing on bone regeneration. Polym Adv Technol2024; 35(1): e6258.
14.
SuXJiaSWangX, et al.Multiple biomaterials for immediate implant placement tissue repair: current status and future perspectives. MedComm–Biomaterials and Applications2024; 3(1): e69.
15.
PinaSReisRLMiguel OliveiraJ. Biocomposites hydrogel-based scaffolds in tissue engineering and regeneration. In: Hydrogels for tissue engineering and regenerative medicine. Cambridge, MA: Academic Press, 2024, pp. 347–365.
16.
ZhangASunLChenK, et al.3D printing viscoelastic hydrogel-based scaffolds with a swelling-dependent gate for cartilage injury regeneration. Chem Eng J2024; 480: 147260.
17.
GuoXLiJWuY, et al.Recent advancements in hydrogels as novel tissue engineering scaffolds for dental pulp regeneration. Int J Biol Macromol2024; 264: 130708.
18.
TamoAKDjouonkepLDWSelabiNBS. 3D printing of polysa/ccharide-based hydrogel scaffolds for tissue engineering applications: a review. Int J Biol Macromol2024; 270: 132123.
19.
DasSValoorRRatnayakeP, et al.Low-concentration gelatin methacryloyl hydrogel with tunable 3D extrusion printability and cytocompatibility: exploring quantitative process science and biophysical properties. ACS Appl Bio Mater2024; 7(5): 2809–2835.
20.
DasSJegadeesanJTBasuB. Advancing peripheral nerve regeneration: 3D bioprinting of GelMA-based cell-laden electroactive bioinks for nerve conduits. ACS Biomater Sci Eng2024; 10(3): 1620–1645.
21.
AmiryaghoubiNFathiMSafaryA, et al.In situ forming alginate/gelatin hydrogel scaffold through schiff base reaction embedded with curcumin-loaded chitosan microspheres for bone tissue regeneration. Int J Biol Macromol2024; 256: 128335.
22.
ChayanunSSoufivandAAFaberJ, et al.Reinforcing tissue-engineered cartilage: nanofibrillated cellulose enhances mechanical properties of alginate dialdehyde–gelatin hydrogel. Adv Eng Mater2024; 26(4): 2300641.
23.
BastamiFSafaviS-MSeifiS, et al.Addition of bone-marrow mesenchymal stem cells to 3D‐printed alginate/gelatin hydrogel containing freeze-dried bone nanoparticles accelerates regeneration of critical size bone defects. Macromol Biosci2024; 24(3): 2300065.
24.
MurphySVAtalaA. 3D bioprinting of tissues and organs. Nat Biotechnol2014; 32(8): 773–785.
25.
KangH-WLeeSJKoIK, et al.A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol2016; 34(3): 312–319.
26.
MajumderNMishraAGhoshS. Effect of varying cell densities on the rheological properties of the bioink. Bioprinting2022; 28: e00241.
27.
Gungor-OzkerimPSInciIZhangYS, et al.Bioinks for 3D bioprinting: an overview. Biomater Sci2018; 6(5): 915–946.
28.
WulandariWTRochliadiAArcanaIM. Nanocellulose prepared by acid hydrolysis of isolated cellulose from sugarcane bagasse. IOP Conf Ser Mater Sci Eng2016; 107(1): 012045.
29.
ListyandaRFWaziz WildanMMochammadNI. Preparation and characterization of cellulose nanocrystal extracted from ramie fibers by sulfuric acid hydrolysis. Heliyon2020; 6(11): e05486.
30.
SchwabALevatoRD’EsteM, et al.Printability and shape fidelity of bioinks in 3D bioprinting. Chem Rev2020; 120(19): 11028–11055.
31.
ChoeGOhSSeokJM, et al.Graphene oxide/alginate composites as novel bioinks for three-dimensional mesenchymal stem cell printing and bone regeneration applications. Nanoscale2019; 11(48): 23275–23285.
32.
MurabSGuptaAWłodarczyk-BiegunMK, et al.Alginate based hydrogel inks for 3D bioprinting of engineered orthopedic tissues. Carbohydr Polym2022; 296: 119964.
33.
WeiQZhouJAnY, et al.Modification, 3D printing process and application of sodium alginate based hydrogels in soft tissue engineering: a review. Int J Biol Macromol2023; 232: 123450.
34.
SaravanouSFIoannidisKDimopoulosA, et al.Dually crosslinked injectable alginate-based graft copolymer thermoresponsive hydrogels as 3D printing bioinks for cell spheroid growth and release. Carbohydr Polym2023; 312: 120790.
35.
HuXZhangZWuH, et al.Progress in the application of 3D-printed sodium alginate-based hydrogel scaffolds in bone tissue repair. Biomater Adv2023; 152: 213501.
36.
LeeSChoiGYangYJ, et al.Visible light-crosslinkable tyramine-conjugated alginate-based microgel bioink for multiple cell-laden 3D artificial organ. Carbohydr Polym2023; 313: 120895.
37.
LiuYHuQDongW, et al.Alginate/gelatin‐based hydrogel with soy protein/peptide powder for 3D printing tissue‐engineering scaffolds to promote angiogenesis. Macromol Biosci2022; 22(4): 2100413.
38.
ShuklaPMitrukaMPatiF. The effect of the synthetic route on the biophysiochemical properties of methacrylated gelatin (GelMA) based hydrogel for development of GelMA-based bioinks for 3D bioprinting applications. Materialia2022; 25: 101542.
39.
GöcklerTHaaseSKempterX, et al.Tuning superfast curing thiol‐norbornene‐functionalized gelatin hydrogels for 3D bioprinting. Adv Healthcare Mater2021; 10(14): 2100206.
40.
NgWLYee YeongWMayWN. Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering. International J of Bioprint2016; 2(1): 53–62.
41.
LiLQinSPengJ, et al.Engineering gelatin-based alginate/carbon nanotubes blend bioink for direct 3D printing of vessel constructs. Int J Biol Macromol2020; 145: 262–271.
42.
ShehzadAMukashevaFMoazzamM, et al.Dual-crosslinking of gelatin-based hydrogels: promising compositions for a 3D printed organotypic bone model. Bioengineering2023; 10(6): 704.
43.
CurtiFDrăgușinD-MSerafimA, et al.Development of thick paste-like inks based on superconcentrated gelatin/alginate for 3D printing of scaffolds with shape fidelity and stability. Mater Sci Eng C2021; 122: 111866.
44.
DiGMLawNWebbB, et al.Mechanical behaviour of alginate-gelatin hydrogels for 3D bioprinting. J Mech Behav Biomed Mater2018; 79: 150–157.
45.
LiZHuangSLiuY, et al.Tuning alginate-gelatin bioink properties by varying solvent and their impact on stem cell behavior. Sci Rep2018; 8(1): 8020.
46.
DasSBasuB. Extrusion‐based 3D printing of gelatin methacryloyl with nanocrystalline hydroxyapatite. Int J Appl Ceram Technol2022; 19(2): 924–938.
47.
YaoBHuTCuiX, et al.Enzymatically degradable alginate/gelatin bioink promotes cellular behavior and degradation in vitro and in vivo. Biofabrication2019; 11(4): 045020.
48.
HabibASathishVMallikS, et al.3D printability of alginate-carboxymethyl cellulose hydrogel. Materials2018; 11(3): 454.
49.
KakarlaABKongITurekI, et al.Printable gelatin, alginate and boron nitride nanotubes hydrogel-based ink for 3D bioprinting and tissue engineering applications. Mater Des2022; 213: 110362.
50.
HuWWangZXiaoY, et al.Advances in crosslinking strategies of biomedical hydrogels. Biomater Sci2019; 7(3): 843–855.
51.
DuttaSDHexiuJPatelDK, et al.3D-printed bioactive and biodegradable hydrogel scaffolds of alginate/gelatin/cellulose nanocrystals for tissue engineering. Int J Biol Macromol2021; 167: 644–658.
52.
WuYLinZYWWengerAC, et al.3D bioprinting of liver-mimetic construct with alginate/cellulose nanocrystal hybrid bioink. Bioprinting2018; 9: 1–6.
53.
MohantySLarsenLBTrifolJ, et al.Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds. Mater Sci Eng C2015; 55: 569–578.
54.
JaiswalAKDhumalRVBellareJR, et al.In vivo biocompatibility evaluation of electrospun composite scaffolds by subcutaneous implantation in rat. Drug Deliv Transl Res2013; 3: 504–517.
55.
BhaskarNBasuB. Osteogenesis, hemocompatibility, and foreign body response of polyvinylidene difluoride-based composite reinforced with carbonaceous filler and higher volume of piezoelectric ceramic phase. Biomaterials2023; 297: 122100.
56.
ChandorkarYBhaskarNMadrasG, et al.Long-term sustained release of salicylic acid from cross-linked biodegradable polyester induces a reduced foreign body response in mice. Biomacromolecules2015; 16(2): 636–649.
57.
BasseLRaskovHHHjort JakobsenD, et al.Accelerated postoperative recovery programme after colonic resection improves physical performance, pulmonary function and body composition. Br J Surg2002; 89(4): 446–453.
58.
Sajad DaneshiSTayebiLTalaei-KhozaniT, et al.Reconstructing critical-sized mandibular defects in a rabbit model: enhancing angiogenesis and facilitating bone regeneration via a cell-loaded 3D-printed hydrogel–ceramic scaffold application. ACS Biomater Sci Eng2024; 10(5): 3316–3330.
59.
BahatibiekeAWeiSFengH, et al.Injectable and in situ foaming shape-adaptive porous bio-based polyurethane scaffold used for cartilage regeneration. Bioact Mater2024; 39: 1–13.
60.
DivakarPMoodieKLDemidenkoE, et al.Quantitative evaluation of the in vivo biocompatibility and performance of freeze-cast tissue scaffolds. Biomed Mater2020; 15(5): 055003.
61.
OsorioMCañasAPuertaJ, et al.Ex vivo and in vivo biocompatibility assessment (blood and tissue) of three-dimensional bacterial nanocellulose biomaterials for soft tissue implants. Sci Rep2019; 9(1): 10553.
62.
JammalMPDa SilvaAAFilhoAM, et al.Immunohistochemical staining of tumor necrosis factor-α and interleukin-10 in benign and malignant ovarian neoplasms. Oncol Lett2015; 9(2): 979–983.
63.
de Souza DantasOSHde Souza AarãoTLda SilvaBL, et al.Immunohistochemical analysis of the expression of TNF-alpha, TGF-beta, and caspase-3 in subcutaneous tissue of patients with HIV lipodystrophy syndrome. Microb Pathog2014; 67: 41–47.
64.
ÖhlingerWPeter DingesHZatloukalK, et al.Immunohistochemical detection of tumor necrosis factor-alpha, other cytokines and adhesion molecules in human livers with alcoholic hepatitis. Virchows Arch1993; 423: 169–176.
65.
al HairTHadisaputraWSoebijantoS, et al.Apoptotic pathways and anti-müllerian hormone affects the ovarian tissue damage during endometrioma cystectomy. Journal of Angiotherapy2024; 8(5): 1–18.
66.
KondoTTakahashiMYamasakiG, et al.Immunohistochemical analysis of CD31 expression in myocardial tissues from autopsies of patients with ischemic heart disease. Leg Med2022; 59: 102127.
67.
PusztaszeriMPSeelentagWBosmanFT. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J Histochem Cytochem2006; 54(4): 385–395.
DasSoumitraRemya Valoor, Jeyapriya Thimukonda Jegadeesan, and Bikramjit Basu. 3D bioprinted GelMA scaffolds for clinical applications: promise and challenges.Bioprinting2024.
70.
DasSoumitraBikramjit Basu. An overview of hydrogel-based bioinks for 3D bioprinting of soft tissues.journal of the Indian Institute of Science2019; 99: 405–428.
71.
IratwarSandeepSulob Roy Chowdhury, Shweta Pisulkar, Soumitra Das, Akhilesh Agarwal, Ashutosh Bagde, Balaji Paikrao, Syed Quazi, and Bikramjit Bas. Comprehensive functional outcome analysis and importance of bone remodelling on personalized cranioplasty treatment using Poly (methyl methacrylate) bone flaps. Journal of Biomaterials Applications2024; 38(no.9): 975–988.
72.
JeyapriyaT.JSulob Roy Choudhury, Bikramjit Basu, Ashutosh Bagde, and Quazi Zahiruddin. Cranioplasty treatment for human healthcare. Current Science2022; 5.
73.
SetareyiRasoolAshrafalsadat Hatamian-Zarmi, Zahra-Beagom Mokhtari-Hosseini, Soheil Kianirad, Ehsan Heidarian, Samira Abbasi-Malati, Narjes Feizollahi, and Mohammad Naji. Biofabrication and evaluation of 3D printed and cast PCL/collagen-alginate hydrogel tubular scaffolds for urethral tissue engineering. International Journal of Biological Macromolecules2025; 142143.
