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Hierarchical helical carbon nanotube fibre as a bone-integrating anterior cruciate ligament replacement

Nature Nanotechnology volume 18, pages 1085–1093 (2023)Cite this article

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Abstract

High rates of ligament damage require replacements; however, current synthetic materials have issues with bone integration leading to implant failure. Here we introduce an artificial ligament that has the required mechanical properties and can integrate with the host bone and restore movement in animals. The ligament is assembled from aligned carbon nanotubes formed into hierarchical helical fibres bearing nanometre and micrometre channels. Osseointegration of the artificial ligament is observed in an anterior cruciate ligament replacement model where clinical polymer controls showed bone resorption. A higher pull-out force is found after a 13-week implantation in rabbit and ovine models, and animals can run and jump normally. The long-term safety of the artificial ligament is demonstrated, and the pathways involved in integration are studied.

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Fig. 1: Hierarchical structure of native ligament and an HHF.
Fig. 2: ACL reconstruction using an HHF in a rabbit model.
Fig. 3: Biointegration of HHF grafts and controls.
Fig. 4: HHF promotes osteogenesis.
Fig. 5: ACL reconstruction using HHF graft restores mobility in a large animal (sheep) model.

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Data availability

Source data are available for Figs. 2 and 3 and Extended Data Figs. 1, 2, 4 and 5 in the associated source data files. The transcriptomics raw data have been deposited in NODE (https://www.biosino.org/node) under accession number OEP002944. All raw data of mass spectrum have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD032128. Source data are provided with this paper.

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Acknowledgements

We thank P. Liu of NAMSA China for support with the sheep experiment, Q. Jin and H. Zhou of Shanghai Institute of Traumatology & Orthopaedics for technical support with the histology preparation, Y. Xu of Fudan University, C. Liu of Shanghai University, Z. Nie of Fudan University and K. Lv of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine for insightful discussions and suggestions, and A.L. Chun of Science Storylab for critically reading and editing the manuscript. This work was supported by the Science and Technology Commission of Shanghai Municipality 20JC1414902 (H.P.), 21511104900 (H.P.), 19441901600 (S.C.), 19441902000 (S.C.); the National Natural Science Foundation of China 22175042 (P.C.), T2222005 (P.C.), 52122310 (X.S.), 22075050 (X.S.), 81572108 (S.C.), 81772339 (S.C.), 11872150 (F.X.), 12122204 (F.X.), 31770886 (C.D.), 31972933 (C.D.), 31700682 (C.D.); the Ministry of Science and Technology of the People’s Republic of China 2017YFA0505102 (C.D.), 2020YFE0201600 (C.D.), 2022YFA1303200 (C.D.); the Shanghai Shuguang Program 21SG05 (F.X.), 19SG02 (C.D.); and the Shanghai Sailing Program 20YF1404500 (L.L.).

Author information

Author notes

  1. These authors contributed equally: Liyuan Wang, Fang Wan, Yifan Xu, Songlin Xie, Tiancheng Zhao, Fan Zhang.

Authors and Affiliations

  1. State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science and Laboratory of Advanced Materials, Fudan University, Shanghai, China

    Liyuan Wang, Yifan Xu, Songlin Xie, Tiancheng Zhao, Han Yang, Jingming Gao, Xiang Shi, Chuang Wang, Xiaoye Yu, Xuemei Sun, Jiandong Ding, Peining Chen & Huisheng Peng

  2. Department of Orthopedic Sports Medicine, Huashan Hospital, The Sports Medicine Institute, Fudan University, Shanghai, China

    Fang Wan & Shiyi Chen

  3. State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Institute of Biomedical Sciences, Human Phenome Institute, Zhongshan Hospital, Fudan University, Shanghai, China

    Fan Zhang, Jiajun Zhu & Chen Ding

  4. Department of Integrative Medicine, Huashan Hospital, The Academy of Integrative Medicine, Fudan University, Shanghai, China

    Linwei Lu

  5. Department of Aeronautics and Astronautics, Fudan University, Shanghai, China

    Yifan Yang & Fan Xu

  6. Vision Research Laboratory, School of Life Sciences, State Key Laboratory of Medical Neurobiology, Collaborative Innovation Centre for Brain Science, Fudan University, Shanghai, China

    Hongbo Yu

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Contributions

Conceptualization: H.P., P.C., X.S. Materials preparation: Y.X., T.Z., H.Y., S.X., L.L., L.W. Animal experiments: F.W., L.W., T.Z., S.X., Y.X. Cell experiments: S.X., J.G., X.Y., H.Y. Transcriptomic and proteomic analysis: F.Z., J.Z., L.W. μCT and histological characterization: Y.X., F.W., S.X. Finite-element simulation: F.X., Y.Y. Schematic diagrams and videos: L.W., C.W., X.S., T.Z. Writing—original draft: L.W., S.X., P.C., H.P., Y.X., F.W. Writing—review and editing: H.P., P.C., X.S., C.D., S.C., J.D., H.Y. Supervision: H.P., C.D., P.C., X.S., S.C.

Corresponding authors

Correspondence to Shiyi Chen, Xuemei Sun, Peining Chen, Chen Ding or Huisheng Peng.

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The authors declare no competing interests.

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Nature Nanotechnology thanks Hala Zreiqat, Georg Duda and Yunzhi Peter Yang for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The mechanical properties of CNT fibres.

a, Photograph showing rolls of continuous CNT fibres. b, c, Photographs showing high strength and light weight of the CNT fibre, respectively. d, Typical specific strength-strain curve of a CNT fibre. e, Mechanical performance of CNT fibre compared to other fibre materials such as metals, polymers and carbon fibres. σ is breaking strength, E is Young’s modulus, and ρ is density. f, Tensile tests performed on CNT fibres show they display stable breaking strengths (σ) after 10,000, 100,000 and 1,000,000 bending cycles. The CNT fibres with length of 1 cm are bent with a curvature radius of 0.247 cm. Tensile tests are performed (with an initial applied stress of 0 MPa) by changing the displacement to determine the breaking strength (σ) of the CNT fibres. Breaking strength after bending is normalized to breaking strength before bending (σ0). n = 4 independent samples. Each point represents an independent measurement. All data are expressed as mean ± s.d. g, h, Photographs showing high flexibility of the CNT fibre under twisting (g) and looping (h). i, j, SEM images of a knotted CNT fibre at low and high magnifications, respectively. k, A Chinese knot formed by the CNT fibre.

Source data

Extended Data Fig. 2 The biocompatibility of HHF.

a, SEM images of MSCs cultured on the control glass group and CNT sheets for 5 days. b, F-actin labelled immunostaining images of the control glass group and CNT sheets after culturing MSCs for 5 days. Red: F-actin; blue: cell nucleus. c, d, F-actin labelled immunostaining image (c) and SEM image (d) of HHF after culturing MSCs for 5 days. Red: F-actin; blue: cell nucleus. e, f, H&E (e) and picrosirius red (f) staining show HHF graft integrates well with bone and skeletal muscle after HHF implanted in rabbits for 13 weeks. g-j, Concentrations of typical plasma markers in rabbit serum after HHF implantation, showing the high biocompatibility of HHF in molecular level. Alanine aminotransferase (ALT) (g), interferon-β (IFN-β) (h), interleukin-1 (IL-1) (i), and tumour-necrosis factor-α (TNF-α) (j) concentrations varied within their normal physiological ranges. Concentrations of plasma markers are determined using enzyme-linked immunosorbent assay. Each point represents the concentration of plasma markers in each rabbit. n = 5 biologically independent experiments for each group. All data are expressed as mean ± s.d.

Source data

Extended Data Fig. 3 Histological analysis to show high structural stability of HHF graft in vivo.

a, H&E sections of cardiac muscle, liver, spleen, lung and kidney after 13 weeks of HHF implantation show no CNT residues. Control: rabbits without HHF. b, Masson’s trichrome-stained tissue sections of cardiac muscle, liver, spleen, lung and kidney after 13 weeks of HHF implantation do not show any collagen deposition and peribronchial lymphocyte aggregation in any of the organs. Control: rabbits without HHF. Red: cytoplasm, muscle and erythrocytes; blue: collagen. c, F4/80 immunostaining of cardiac muscle, liver, spleen, lung and kidney after HHF implanted in rabbits for 13 weeks do not show any inflammatory cell infiltration in any of the organs. Control: rabbits without HHF. Nucleus is stained with DAPI (blue); inflammatory cells are stained by both DAPI and F4/80 antibodies (blue and green).

Extended Data Fig. 4 ACL reconstruction using HHFs and controls in a rabbit model.

a, μCT images of the femur and tibia of rabbits after surgery for 4 and 13 weeks with HHF and PET fibres as the ACL grafts. b, Three-dimensional images rebuilt from μCT data of the femur and tibia of rabbits after the surgery for 4 and 13 weeks with carbon fibres as the ACL graft. c, Average diameters, ratios of bone surface (BS) to total volume (TV) and bone volume (BV) to TV of femoral and tibia tunnels of rabbits after implanting carbon fibres for 4 and 13 weeks. n = 5 biologically independent experiments for each group. All data are expressed as mean ± s.d. d, Photographs of the femur-graft-tibia complex specimens of rabbits after the surgery for 13 weeks with HHF, PET fibres and carbon fibres as the ACL grafts.

Source data

Extended Data Fig. 5 Bio-integration of HHF grafts and controls.

a, Force-strain curves of pull-out tests for native ligament, HHF, carbon fibre and PET fibre after implantation for 13 weeks. b, SEM images showing the surface of the pull-out HHF and PET fibre implants after 13 weeks of implantation. c, Immunostaining and SEM images of osteoblasts cultured on the CNT sheets and PET fibres for 5 days, respectively. d, Photographs showing the moving trial of the right hindleg with ACL torn (up) and reconstructed using the grafts of HHF (below) during one hopping process. e, H&E staining of the bone tunnel after the surgery for 13 weeks with PET fibre and carbon fibre as the ACL graft. f, Picrosirius-red-stained slices of bone tunnel implanted with carbon fibre under bright field (left) and polarized light (right) show almost no collagen at the carbon fibre/host bone interface.

Source data

Extended Data Fig. 6 Histological analysis to show the activity of osteoblasts, re-established crimp, re-vascularization and re-innervation induced by HHF implanted in rabbits.

a, Methylene blue-acid fuchsin-stained slices of bone tunnel after HHF implantation in rabbits. Dark blue: osteoblasts; purple grey: osteoid tissue; red: newly formed bone. b, Masson Goldner-stained slices of bone tunnel after HHF implantation in rabbits. Red: mature bone; green: collagen; red-green: newly formed bone. c, Picrosirius-red-stained slices of bone tunnel implanted with HHF under bright field. Collagen tissues are gradually formed at the interfacial region between HHF and host bone from Week 1 to Week 4. d, (i) VEGF-stained images. Nucleus is stained with DAPI (blue); vessels are stained by both DAPI and VEGF antibodies (blue and red). The position of HHF is marked with white pentalpha. (ii) Magnified view of yellow box in (i) shows VEGF-positive vessels are formed at the interface of HHF/native bone. e, (i) CGRP-stained images. Nucleus is stained with DAPI (blue); nerves are stained by both DAPI and CGRP antibodies (blue and green). The position of HHF is marked with white pentalpha. (ii) Magnified view of yellow box in (i) shows CGRP-positive nerves are formed at the interface of HHF/native bone.

Extended Data Fig. 7 ACL reconstruction in a rabbit model after 18 months of HHFs and PET fibres implantation.

a, μCT images showing a coronal section of a femoral tunnel (top) and an axial section of a tibial tunnel (bottom, yellow arrow) after 18 months of HHF and PET fibre implantation, showing new bone tissues are completely occupied in the tunnel between the host bone and HHF. b, H&E-stained images of rabbit joint section after 18 months of HHF implantation show dense tissue around HHF (black) and good integration of HHF with surrounding bone. c, Picrosirius-red-stained slices of rabbit joint implanted with HHF for 18 months under bright field (left) and polarized light (right) show newly formed bones inside the channels among the primary fibres of HHF. A thick layer of anisotropic collagen (green) around the primary fibres of HHF is formed.

Extended Data Fig. 8 HHF promotes osteogenesis in vitro and in vivo by activating osteogenesis-related signalling pathways.

a, MSCs cultured on HHF express higher levels of typical osteogenesis-related genes than those cultured on helical PET fibre. Horizontal dotted lines represent mean of helical PET fibre group. n = 5 biologically independent experiments for each group. All data are expressed as mean ± s.d. Statistical significance was determined by unpaired two-tailed t-test (n.s., not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001). b, GO and KEGG enrichment based on transcriptomic analysis of MSCs cultured on HHF and helical PET fibre show upregulated osteogenesis-related signalling pathways. n = 5 biologically independent experiments for each group. c, SEM images of different substrates for MSC culturing. (i) PET plate. (ii) Helical PET fibre. (iii) Helical carbon fibre. (iv) HHF. d, Heatmap analysis of differentially expressed genes related to osteogenesis of MSCs cultured on PET plate, helical PET fibre, helical carbon fibre and HHF. n = 5 biologically independent experiments for each group. e, MSCs cultured on PET plate, helical PET fibre, helical carbon fibre and HHF express different levels of typical osteogenesis-related genes in MAPK (up) and WNT (below) signalling pathway. Horizontal dotted lines represent mean of PET plate group. n = 5 biologically independent experiments for each group. Data are expressed as mean ± s.d. Statistical significance was determined by unpaired two-tailed t-test between two groups and one-way ANOVA among multiple groups (n.s., not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.005 and ****P < 0.001). f, GO and KEGG enrichment based on transcriptomic analysis of MSCs show upregulated osteogenesis-related signalling pathways for different pairwise comparisons (carbon component: helical carbon fibre vs helical PET fibre; micrometre channel: helical PET fibre vs PET plate; nanometre channel: HHF vs helical carbon fibre). n = 5 biologically independent experiments for each group. g, GO and KEGG enrichment based on proteomic analysis of rabbit bone implanted with HHF and helical PET fibre show osteogenesis-related signalling pathways are upregulated in vivo. n = 3 biologically independent experiments for each group. Pathway enrichment analysis in (b, f, g) was performed by DAVID (https://david.ncifcrf.gov/) and ConsensusPathDB (http://cpdb.molgen.mpg.de/CPDB), P value is calculated based on the hypergeometric distribution and the significant pathway (P value < 0.05) was used for further study.

Extended Data Fig. 9 ACL reconstruction using HHF graft restores mobility in a large animal model of sheep.

a, CT images of tibias in a coronal plane using HHFs and PET fibres as grafts after surgery for 13 weeks. The tunnels are marked with yellow dashed lines. b, CT images of the tibia scanned in axial plane at the position marked with blue lines in (a). c, Photograph of the postoperative sheep standing with a normal gait in Week 13. d, H&E-stained image of the tibial tunnels after 13 weeks of implanting HHFs. e, Enlarged view of yellow box marked in (d). The newly-formed bone tissues are observed in the channels within the HHF. f, Picrosirius-red-stained slices of the bone tunnels after implanting HHFs for 13 weeks.

Supplementary information

Supplementary Information

Supplementary Notes 1–9, Figs. 1–24, Tables 1 and 2, captions for Supplementary Movies 1–4, captions for Supplementary Data 1–7, and references.

Reporting Summary

Supplementary Table 1

Differentially expressed proteins and genes related to osteogenesis of MSCs cultured on different substrates.

Supplementary Video 1

Movement of a rabbit with reconstructed ACL using HHF graft on the hindlimb.

Supplementary Video 2

Movement of a control rabbit with torn ACL in the hindlimb.

Supplementary Video 3

Movement of a sheep with reconstructed ACL using HHF graft after 13 weeks of implantation.

Supplementary Video 4

Movement of a sheep with reconstructed ACL using HHF graft after 9 months of implantation.

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Wang, L., Wan, F., Xu, Y. et al. Hierarchical helical carbon nanotube fibre as a bone-integrating anterior cruciate ligament replacement. Nat. Nanotechnol. 18, 1085–1093 (2023). https://doi.org/10.1038/s41565-023-01394-3

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