Research ArticleMEDICAL ROBOTS

Human adipose–derived mesenchymal stem cell–based medical microrobot system for knee cartilage regeneration in vivo

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Science Robotics  22 Jan 2020:
Vol. 5, Issue 38, eaay6626
DOI: 10.1126/scirobotics.aay6626
  • Fig. 1 Concept overview of knee cartilage regeneration procedures using magnetic microrobot-mediated MSC delivery system.

    The magnetic microrobot containing MSCs was prepared through a sequential process by adsorption of magnetic microclusters on the PLGA microscaffold and MSCs loading (step 1). The prepared MSC-loaded microrobots were delivered to cartilage defect using EMA system (step 2). After the targeted delivery procedure, the microrobots are immobilized to the defect using a permanent magnet (step 3).

  • Fig. 2 Characterization of magnetic microrobot and microcluster.

    (A) SEM image of a magnetic microcluster. (B) Size distribution of magnetic microclusters in deionized water. (C) Zeta potential graph showing ionic surface charge of ferumoxytol and magnetic microparticle. (D) SEM images of magnetic microrobots. Top and bottom: Overall 3D porous structure and pore morphology of the microrobot, respectively. (E) EDX mapping of microrobot, which is mainly composed of carbon, oxygen, and iron. (F) Size distribution of microrobots and their pores. (G) FTIR spectra of chitosan, ferumoxytol, magnetic microcluster, PLGA microscaffold, and microrobot. a.u., arbitrary units. (H) TGA curves of magnetic microcluster, PLGA microscaffold, and microrobot. (I) Optical microscopy and SEM images of microrobots treated with lysozyme solution. On day 33, the scale bars of upper and lower SEM images are 100 and 20 μm, respectively. (J) Changes in diameter of microrobots with lysozyme solution with respect to time (n > 13). (K) Photographs showing responses of microrobots to physical shaking and magnetic attraction. (L) Magnetic hysteresis curves of ferumoxytol, magnetic microcluster, and microrobot measured by VSM.

  • Fig. 3 Microrobots do not affect differentiation potency and provide a suitable environment for cell growth.

    (A) Evaluation of cytotoxicity after ferumoxytol (0 to 64 μg ml−1) and magnetic microcluster treatment for 24 hours (n = 3; *P < 0.05, Student’s t test). (B) Evaluation of cytotoxicity after cell culture in PLGA microscaffold and microrobot for 24 hours (n = 4). (C) Number of cells attached to the microrobot according to the incubation time after seeding 5000 cells into each microrobot. (D) Confocal images of hADMSC-microrobot, in which MSCs were incubated in the microrobot for 24 hours. Red and blue represent cytoplasmic and nuclear staining, respectively. (E) hADMSC proliferation in microrobot and PLGA-microscaffold for 1 to 22 days (n = 3). (F) Image of differentiated hADMSC-microrobot after culturing for 21 days in chondrogenic differentiation medium. Red and blue depict the expression of COLII and the nuclei, respectively. (G) Expression of cartilage-specific genes after 21 days (n = 3; *P < 0.05 and **P < 0.01, Student’s t test).

  • Fig. 4 EMA system with optimized coil figuration provides high targeting efficiency of microrobot.

    (A) Photograph showing the EMA system combined with imaging devices. (B) Mobility of microrobot at different magnetic fields and gradients (n > 3). (C) Mobility change of microrobot according to cell loading (n = 4). (D) Schematic diagram of a 3D targeting test in different phantoms (left). Screenshots of movies (right) show targeting of microrobot with and without EMA system at hole B. (E) Targeting efficiency of microrobots at each hole (n > 5; ***P < 0.001, Student’s t test). (F) Time-lapse image sequence of microrobots during targeting at defect of medial condyle (left) and patella (right) in ex vivo porcine knee. In each image, the red arrows in knee joint model indicate the defect position. The time is indicated on each image in the minutes:seconds format.

  • Fig. 5 Magnet enhancing MSC transport to cartilage defect through magnetic fixation of microrobots.

    (A) Schematic illustration of magnet placement on rabbit knee joint. Magnetic fields and gradient maps inside black dashed-line box indicate numerical simulation results of cartilage defect. The graph inside blue dashed-line box shows optimization of magnet size. (B) Numerical simulation results of magnetic fields generated from a magnet within defect region. In each magnetic field map, three red points on dotted lines (A, B, and C) were used for comparison of measured and simulated magnetic fields. (C) Simulated (A-s, B-s, and C-s) and measured (A-m, B-m, and C-m) magnetic fields at 45 positions within the defect region. (D) Time-lapse images of microrobot fixation test performed without and with the magnet. Areas near the numbered arrows are enlarged in the insets. Scale bars, 3 mm. Time is indicated on each image in the minutes:seconds format. (E) Fixation rates of microrobots without (−) and with (+) the magnet (n = 3; ***P < 0.001, Student’s t test). (F) Adhesion rates of microrobots according to cell loading, use of magnet, and cell culture day (n = 3; *P < 0.05, Student’s t test). MR, microrobot; hMR, hADMSC-microrobot; ns, not significant.

  • Fig. 6 In vivo cartilage regeneration using magnetic microrobot-mediated MSC delivery system.

    (A) Images of EMA system and magnet applied to rabbit knee in targeting (top) and fixation (bottom) steps. (B) Macroscopic appearances of microrobots after magnetic targeting (top) and 1 week after magnetic fixation (bottom). Areas near the numbered arrows are displayed in the insets. Scale bars, 2 mm. (C) Image of gross (top) and Prussian blue staining (bottom) at the first week and 3 weeks after hADMSC-microrobot injection. The green (left) and yellow (right) dotted squares represent the Prussian blue iron staining area at the first week and 3 weeks, respectively. (D) Fluorescence image of engrafted cells present in cartilage using CellTrace (red) 3 weeks after hADMSC-microrobot injection. DAPI (blue) was used for counterstaining of CellTrace. (E) Expression of inflammatory genes IL1β, IL-6, TNF-α, and IL-8 in tissue isolated from inflammatory response and regulatory organs after microrobot injection (n = 4). (F) H&E and COLII staining of the cartilage tissue in defect group (top) and microrobot system group (w/o, middle; w/, bottom) at 3 weeks. (G) COLII expression quantified by ImageJ software (n = 3; **P < 0.01, Student’s t test).

Supplementary Materials

  • robotics.sciencemag.org/cgi/content/full/5/38/eaay6626/DC1

    Materials and Methods

    Section S1. Materials of the microrobot

    Section S2. Fabrication of the microrobot

    Section S3. Actuation principles of the microrobot

    Section S4. Control principle and optimal coil placement of the EMA system

    Section S5. Characterization and calibration of the EMA system

    Section S6. Experimental setup for microrobot targeting tests using the EMA system

    Section S7. Experimental setup for fixation and adhesion tests of the microrobot

    Section S8. Cell viability assay

    Section S9. Evaluation of cell proliferation and loading of the microrobot

    Section S10. Chondrogenic differentiation

    Section S11. Reverse transcription polymerase chain reaction and real-time polymerase chain reaction

    Section S12. Animals

    Section S13. Evaluation of inflammatory responses to PLGA microscaffold and microrobot

    Section S14. Histological and immunohistochemical analysis

    Fig. S1. SEM images of PLGA microscaffolds after w-o-w emulsion–based gelatin leaching.

    Fig. S2. PLGA microscaffold does not affect chondrogenic differentiation.

    Fig. S3. Cartilage-specific gene expression gradually increases with the period of chondrogenic differentiation.

    Fig. S4. Phantom with patella and medial condyle defect models for microrobot targeting test using a single magnet.

    Fig. S5. Microrobot targeting with and without magnet at patella and medial condyle defect models.

    Fig. S6. Experimental setup of EMA system and arthroscope for ex vivo targeting test on the porcine knee joint.

    Fig. S7. Microrobot mobility in aqueous solution with different viscosity.

    Fig. S8. Numerical simulation result of fluid flow by different directions of microrobot injection.

    Fig. S9. Time-lapse image sequence of microrobots without magnetic targeting at the defect of medial condyle (left) and patella (right) in ex vivo porcine knee.

    Fig. S10. A single magnet helps magnetic fixation of microrobot located at patella cartilage defects.

    Fig. S11. Illustration and numerical simulation of microrobot fixation for medial condyle defects using the magnet placed on tibia.

    Fig. S12. Numerical simulation results of magnetic field gradient generated from a magnet in defect region.

    Fig. S13. Fluorescence image of engrafted hADMSC in cartilage tissue.

    Fig. S14. Histological analysis of cartilage regeneration by microrobot system.

    Fig. S15. Upper and lower regions of coil placement.

    Fig. S16. First and last coil configurations among coil configuration candidates obtained through the optimization routine.

    Fig. S17. Characterization and calibration of EMA system.

    Fig. S18. Experimental setup for magnetic fixation of microrobot.

    Table S1. Design parameters of the EMA system.

    Table S2. Specifications of the EMA system with optimal coil configuration.

    Table S3. Angle range of coils within the region of coil placement (fig. S15).

    Table S4. Comparison between first and last coil configurations (fig. S16) for the coil configuration candidate obtained through the optimization routine.

    Movie S1. Microrobot targeting with and without magnet at patella and medial condyle defect models.

    Movie S2. Manipulation of microrobots in a knee joint phantom.

    Movie S3. 3D magnetic targeting of microrobots in five phantoms with a hole.

    Movie S4. Magnetic targeting of microrobots in ex vivo porcine knee cartilage at medial condyle and patella defects.

    Movie S5. Magnetic fixation of microrobots in ex vivo porcine knee cartilage.

    Movie S6. Adhesion of hADMSC-microrobots in ex vivo porcine knee cartilage.

    Movie S7. Magnetic targeting of hADMSC-microrobots in rabbit knee cartilage with a medial condyle defect.

    References (4852)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Section S1. Materials of the microrobot
    • Section S2. Fabrication of the microrobot
    • Section S3. Actuation principles of the microrobot
    • Section S4. Control principle and optimal coil placement of the EMA system
    • Section S5. Characterization and calibration of the EMA system
    • Section S6. Experimental setup for microrobot targeting tests using the EMA system
    • Section S7. Experimental setup for fixation and adhesion tests of the microrobot
    • Section S8. Cell viability assay
    • Section S9. Evaluation of cell proliferation and loading of the microrobot
    • Section S10. Chondrogenic differentiation
    • Section S11. Reverse transcription polymerase chain reaction and real-time polymerase chain reaction
    • Section S12. Animals
    • Section S13. Evaluation of inflammatory responses to PLGA microscaffold and microrobot
    • Section S14. Histological and immunohistochemical analysis
    • Fig. S1. SEM images of PLGA microscaffolds after w-o-w emulsion–based gelatin leaching.
    • Fig. S2. PLGA microscaffold does not affect chondrogenic differentiation.
    • Fig. S3. Cartilage-specific gene expression gradually increases with the period of chondrogenic differentiation.
    • Fig. S4. Phantom with patella and medial condyle defect models for microrobot targeting test using a single magnet.
    • Fig. S5. Microrobot targeting with and without magnet at patella and medial condyle defect models.
    • Fig. S6. Experimental setup of EMA system and arthroscope for ex vivo targeting test on the porcine knee joint.
    • Fig. S7. Microrobot mobility in aqueous solution with different viscosity.
    • Fig. S8. Numerical simulation result of fluid flow by different directions of microrobot injection.
    • Fig. S9. Time-lapse image sequence of microrobots without magnetic targeting at the defect of medial condyle (left) and patella (right) in ex vivo porcine knee.
    • Fig. S10. A single magnet helps magnetic fixation of microrobot located at patella cartilage defects.
    • Fig. S11. Illustration and numerical simulation of microrobot fixation for medial condyle defects using the magnet placed on tibia.
    • Fig. S12. Numerical simulation results of magnetic field gradient generated from a magnet in defect region.
    • Fig. S13. Fluorescence image of engrafted hADMSC in cartilage tissue.
    • Fig. S14. Histological analysis of cartilage regeneration by microrobot system.
    • Fig. S15. Upper and lower regions of coil placement.
    • Fig. S16. First and last coil configurations among coil configuration candidates obtained through the optimization routine.
    • Fig. S17. Characterization and calibration of EMA system.
    • Fig. S18. Experimental setup for magnetic fixation of microrobot.
    • Table S1. Design parameters of the EMA system.
    • Table S2. Specifications of the EMA system with optimal coil configuration.
    • Table S3. Angle range of coils within the region of coil placement (fig. S15).
    • Table S4. Comparison between first and last coil configurations (fig. S16) for the coil configuration candidate obtained through the optimization routine.
    • Legends for movies S1 to S7
    • References (4852)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Microrobot targeting with and without magnet at patella and medial condyle defect models.
    • Movie S2 (.mp4 format). Manipulation of microrobots in a knee joint phantom.
    • Movie S3 (.mp4 format). 3D magnetic targeting of microrobots in five phantoms with a hole.
    • Movie S4 (.mp4 format). Magnetic targeting of microrobots in ex vivo porcine knee cartilage at medial condyle and patella defects.
    • Movie S5 (.mp4 format). Magnetic fixation of microrobots in ex vivo porcine knee cartilage.
    • Movie S6 (.mp4 format). Adhesion of hADMSC-microrobots in ex vivo porcine knee cartilage.
    • Movie S7 (.mp4 format). Magnetic targeting of hADMSC-microrobots in rabbit knee cartilage with a medial condyle defect.

    Files in this Data Supplement:

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