Research ArticleTissue Engineering

Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration

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Science Translational Medicine  05 Jun 2019:
Vol. 11, Issue 495, eaav7756
DOI: 10.1126/scitranslmed.aav7756
  • Fig. 1 Engineered mesenchymal condensations.

    (A) Photograph of hMSC sheets containing TGF-β1–loaded GMs self-assembled for 2 days on Transwell inserts (left) before combination into engineered mesenchymal condensations for implantation (right). Each condensation was assembled from three sheets and enclosed within a perforated electrospun nanofiber mesh tube of polycaprolactone. (B) Photograph of a critically sized (8 mm) segmental bone defect created in the femora of an RNU rat. Engineered mesenchymal condensations were implanted into bone defects. (C and D) Timelines of in vitro and in vivo analyses. (C) hMSC sheets were evaluated in vitro at 2 days (the time point of transplantation) or at 23 days of culture in chondrogenesis-supportive medium. (D) Bone regeneration, neovascularization, and endochondral ossification were evaluated over 3 to 12 weeks after transplantation. (E) Safranin O/fast green staining of hMSC sheets with empty or 600 ng of TGF-β1–containing GMs, cultured for 2 or 23 days in vitro. Red indicates sGAGs; fast green counterstain shows cells and remaining matrix. (F) mRNA expression of chondrogenic, osteogenic, and YAP pathway genes at days 2 and 23 in vitro; quantitative reverse transcription polymerase chain reaction (qRT-PCR) results normalized to glyceraldehyde-phosphate dehydrogenase (GAPDH) and expressed as fold change over empty microsphere control sheets (n = 3 sheets per group). (G) Immunoblot (IB) of phosphorylated-SMAD3 activity at day 2 in vitro with β-actin control and (H) band intensity of p-SMAD3/SMAD3 ratio expressed as fold change over sheets without growth factor. (I) Immunostaining for YAP and CYR61 at days 2 and 23 in vitro. 3,3ʹ-diaminobenzidine (DAB) staining with peroxidase was used to produce a brown reaction product at locations of immunolabeled antigens. Right: Negative control isotype IgG (rabbit, top; mouse, bottom). *P < 0.05, **P < 0.01, ***P < 0.001 TGF-β1–treated versus empty, unpaired two-tailed Student’s t test for each independent gene. Data shown are means ± SD. Scale bars, 100 μm.

  • Fig. 2 Mechanical loading enhanced endochondral bone regeneration.

    (A) Schematic of fixation plate configurations for dynamic control of ambulatory load transfer. (B) Schematic of loading timeline. Early loading features compliant plate actuation at implantation; delayed loading features unlocking at week 4. (C) Representative in vivo microCT reconstructions at week 4. (D) Safranin O/fast green staining of sagittal histological sections at week 4 (left) in comparison to the native rat distal femur growth plate (right). Bottom row: Magnification of boxed areas. (E) Longitudinal microCT quantification of bone volume at week 4 [n = 11, 11, 9, and 8 for stiff, early, delayed, and BMP-2/collagen (stiff), respectively], week 8 (n = 10, 9, 8, and 8), and week 12 (n = 10, 8, 8, and 8). Repeated significance indicator letters (a, b, and c) signify P > 0.05 (not significant); and groups with distinct indicators (a versus b) signify P < 0.05 at each time point. (F) Representative 3D microCT reconstructions at week 12. (G) Local trabecular thickness mapping on transverse sections, indicated by boxed arrows in (F), in comparison to the native bone of the ipsilateral femoral head (H). (I) MicroCT quantification of trabecular thickness (Tb.Th), number (Tb.N), and spacing (Tb.Sp) in reference to that of the ipsilateral femoral head (femoral head mean ± SD shown as dotted lines and shaded pink region). (J) Hematoxylin and eosin (H&E)–stained histological sections at week 4 (representative sample from n = 1 per group). (K) Representative 3D microCT reconstruction of BMP-1/collagen group at week 12. (L) Trabecular thickness mapping on the section indicated in (K) illustrating heterotopic bone. Scale bars, 100 μm. Data shown with mean ± SEM. **P < 0.01, one- or two-way ANOVA with Tukey’s post hoc analysis.

  • Fig. 3 Transplanted cell function.

    (A) Timeline depicting devitalization of mesenchymal condensations performed at day 2, before transplantation. (B and C) MicroCT analysis of bone formed at week 12 in live and devitalized groups (n = 10 and 5 for live and devitalized, respectively). Dashed lines illustrate the location of the native bone ends. (D) Representative safranin O/fast green staining at the center of the defects at week 12 (n = 1 to 3 per group). Representative samples selected on the basis of mean bone volume. Scale bar, 100 μm. (E) HuNu staining of live and devitalized samples (stiff plates) at week 12. DAB peroxidase produced a brown reaction product at locations of immunolabeled antigens. Dashed lines indicate the edges of the native cortical bone at the distal end of the defect. Devitalized samples exhibited some matrix-associated nonspecific staining, as shown in IgG controls. (F) Immunostaining of HuNU, YAP, and CYR61 in defects of the delayed loading group (live cells). In each case, DAB peroxidase was used to produce a brown reaction product at locations of immunolabeled antigens. Isotype-matched IgG controls were used to demonstrate specificity. Bottom row shows magnification of boxed areas. Scale bars, 100 μm.

  • Fig. 4 Endochondral matrix formation.

    Tile scan images of Safranin O/fast green–stained histological sections of representative samples from stiff, early, and delayed loading groups at (A) week 4 and (B) week 12. Scale bar, 3 mm. All samples oriented distal (left) to proximal (right). Dotted lines in top left indicate the native cortical bone ends. Label “dh” indicates location of fixation plate drill holes. Scale bar, 3 mm. (C) Magnified images of dashed boxed regions in (B) showing endochondral cartilage remnants at week 12. Scale bar, 100 μm. Bottom row: Magnification of boxed regions in the top row. (D) Polarized light microscopy of picrosirius red–stained histological sections at week 12. Increased birefringent intensity indicates increased collagen fibril organization associated with bone matrix remodeling to lamellar bone; reduced birefringence indicates greater amounts of woven bone. Scale bars, 100 μm. Bottom row: Magnification of boxed regions in upper row. All images were taken from a representative sample that most closely matched the average in vivo microCT morphometry of that group.

  • Fig. 5 Restoration of mechanical function.

    Structural mechanical properties were measured by torsion to failure at week 12. Age-matched intact bone properties are shown as dotted lines/gray shading indicating mean ± SD. Samples with full defect bridging are shown in filled data points; open data points indicate nonbridged samples. (A) Analysis of torsional stiffness, (B) maximum torque at failure, (C) minimum pMOI, and (D) average pMOI (n = 8, 7, and 7 for stiff, early, and delayed, respectively). Best subsets regression analysis with lowest AIC value for measured and predicted torsional stiffness (E) and maximum torque at failure (F), indicating significant contributions of minimum pMOI (Jmin) and binary bridging score. Error bars show means ± SD with individual data points. Statistical comparisons between groups for each measure were performed by one-way ANOVA with Tukey’s post hoc analyses, *P < 0.05; †P < 0.05 versus intact bone.

  • Fig. 6 Mechanical control of neovascularization.

    (A) Schematic of stiff versus early loading featuring compliant plate actuation at implantation and contrast agent perfused at week 3. (B and C) Representative microCT reconstructions of bone (B) and blood vessels with local vessel diameter mapping (C) under stiff and early loading conditions at week 3. (D) Quantification of bone volume. (E to H) 3D vascular network morphometry quantifying vascular volume (E), connectivity (F), and vessel orientation and distribution, as measured by degree of anisotropy (G) and the angle with respect to the bone axis of the maximum principal eigenvector (H2) of the mean intercept length (MIL) tensor (H), indicating the dominant direction of vessel orientation. Degree of anisotropy represents the ratio of the longest and shortest MIL eigenvalues; degree of anisotropy = 1 indicates isotropy. (I) Schematic of stiff versus delayed loading featuring compliant plate unlocking at week 4 and contrast agent perfused at week 7. (J to L) Representative microCT reconstructions of bone (J) and blood vessels with local vessel diameter mapping (K) under stiff and early loading conditions at week 7. (L) Quantification of bone volume. (M to P) Vascular network morphometry measured by vascular volume (M), connectivity (N), degree of anisotropy (O), and maximum principal vector (p-vector) angle (P). Quantification is in a 5-mm ROI, paired data are either shown as means ± SEM or superimposed on box plots displaying median as horizontal line, interquartile range as boxes, and minimum/maximum range as whiskers. Mean values are indicated by +. Comparisons between groups were evaluated by paired two-tailed Student’s t tests (*P < 0.05).

  • Fig. 7 Spatial distribution of neovascular and cartilaginous tissues.

    (A) Axial view of 3D neovessel diameter mapping under stiff and early loading conditions at week 3, n = 10. (B) ROI analysis to quantify vascular volume fraction in a 1.5-mm-diameter core region compared to a 5- to 1.5-mm annular region (inset). (C) Cationic (CA4+) cartilage contrast agent–enhanced microCT quantification of cartilage in annulus and core regions. n = 5 to 6. (D) Axial view of 3D neovessel diameter mapping under stiff and delayed loading conditions at week 7, n = 8. (E) ROI analysis of vascular volume fraction. (F) Cartilage contrast agent–enhanced microCT quantification of cartilage in annulus and core regions at week 7, n = 5 to 6. (G) Representative image of coregistered contrast agent–enhanced cartilage with microCT angiography of neovasculature. Cartilage is shaded blue and vessels are red. (H) Safranin O/fast green–stained histological sections of vascular contrast agent–perfused tissues showing the avascular cartilage core and blood vessels in the surrounding tissue (3 weeks). Residual contrast agent exhibits thermal contraction during paraffin processing, visible as dark dots in vessel lumens. Scale bar, 100 μm. Data shown as means ± SEM either with individual data points or with box plots displaying median as horizontal line, interquartile range as boxes, and minimum/maximum range as whiskers. Mean values on box plots are indicated by +. (*P < 0.05, two-way ANOVA with Tukey’s post hoc comparisons)

  • Fig. 8 In vitro analysis of mechanical load on chondrogenic lineage progression.

    (A) Photograph of hMSC-laden hydrogel and schematic of custom-made bioreactor applying dynamic compression. (B) Timeline of the four loading groups evaluated: free swelling (FS) controls, early loading (continuous for 5 weeks), delayed loading (free-swelling for 3 weeks followed by 2 weeks of loading), and reversed loading (loading for 2 weeks followed by 3 weeks of free swelling). (C) Quantification of DNA, sGAG, and total collagen content (n = 5). (D) Alcian blue and (E) pericellular COL6a1 immunostaining. DAB peroxidase was used to produce a brown reaction product at locations of immunolabeled antigen. Images shown at 20× with 10× insets. Scale bars, 100 μm. (F and G) qPCR at week 5 (n = 4 to 5 per group) of (F) COL6a1 and (G) SOX9, COL10a1, OPN, and VEGF. Relative expression was calculated as fold change over free swelling controls. Data are shown as means ± SD with individual data points. Statistical comparisons between groups for each measure were performed by one-way ANOVA with Tukey’s post hoc analyses, where groups sharing a letter (a, b, and c) are not statistically different.

  • Table 1 Oligonucleotide primer sequences for qRT-PCR.

    Fwd, forward; Rev, reverse.

    GeneSequence (5′-3′)Accession number
    SOX9FwdCACACAGCTCACTCGACCTTGNM_000346.3
    RevTTCGGTTATTTTTAGGATCATCTCG
    ACANFwdTGCGGGTCAACAGTGCCTATCNM_001135.3
    RevCACGATGCCTTTCACCACGAC
    COL2A1FwdGGAAACTTTGCTGCCCAGATGNM_001844.4
    RevTCACCAGGTTCACCAGGATTGC
    OSXFwdTGGCTAGGTGGTGGGCAGGGNM_001173467.2
    RevTGGGCAGCTGGGGGTTCAGT
    RUNX2FwdACAGAACCACAAGTGCGGTGCAANM_001015051.3
    RevTGGCTGGTAGTGACCTGCGGA
    ALPFwdCCACGTCTTCACATTTGGTGNM_000478.4
    RevGCAGTGAAGGGCTTCTTGTC
    COL1A1FwdGATGGATTCCAGTTCGAGTATGNM_000088.3
    RevGTTTGGGTTGCTTGTCTGTTTG
    GAPDHFwdGGGGCTGGCATTGCCCTCAANM_002046.5
    RevGGCTGGTGGTCCAGGGGTCT
    YAPFwdCAACTCCAACCAGCAGCAACANM_001130145
    RevGCAGCCTCTCCTTCTCCATCTG
    TAZFwdACCCACCCACGATGACCCCANM_015472
    RevGCACCCTAACCCCAGGCCAC
    CTGFFwdAGGAGTGGGTGTGTGACGANM_001901
    RevCCAGGCAGTTGGCTCTAATC
    CYR61FwdGAGTGGGTCTGTGACGAGGATNM_001554
    RevGGTTGTATAGGATGCGAGGCT
    GAPDHFwdCTTTTGCGTCGCCAGNM_002046
    RevTTGATGGCAACAATATCCAC
    SOX9FwdCTCTGGAGACTTCTGAACGNM_000346
    RevAGATGTGCGTCTGCTC
    ACANFwdTGCGGGTCAACAGTGCCTATCNM_001135
    RevCACGATGCCTTTCACCACGAC
    COL2a1FwdGAAGAGTGGAGACTACTGGNM_033150
    RevCAGATGTGTTTCTTCTCCTTG
    OPN (SPP1)FwdGACCAAGGAAAACTCACTACNM_001251829
    RevCTGTTTAACTGGTATGGCAC
    RUNX2FwdAAGCTTGATGACTCTAAACCNM_001015051
    RevTCTGTAATCTGACTCTGTCC
    COL10a1FwdGCTAGTATCCTTGAACTTGGNM_000493
    RevCCTTTACTCTTTATGGTGTAGG
    VEGFAFwdAATGTGAATGCAGACCAAAGNM_001204384
    RevGACTTATACCGGGATTTCTTG
    COL6a1FwdACAGTGACGAGGTGGAGATCANM_001848
    RevGATAGCGCAGTCGGTGTAGG

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/495/eaav7756/DC1

    Fig. S1. H&E staining of murine embryonic limb buds and hMSC sheets used for preparation of mesenchymal condensations.

    Fig. S2. Fixation plate characterization.

    Fig. S3. Bone accumulation and bridging rates.

    Fig. S4. MicroCT ROI analysis demonstrating formation of cortical and trabecular bone compartments.

    Fig. S5. Full histological analysis of endochondral bone formation at week 4.

    Fig. S6. Full histological analysis of endochondral bone formation at week 12.

    Fig. S7. Collagen organization.

    Fig. S8. Histology of contrast agent–perfused sections.

    Fig. S9. Best subsets analysis of mechanical testing data, models 2 to 5.

    Fig. S10. Vasculature in defect periphery.

    Fig. S11. mRNA expression of hMSCs in dynamically compressed hydrogels.

    Movie S1. Fixation plate actuation and bone regeneration.

    Data file S1. Primary data.

    References (7476)

  • The PDF file includes:

    • Fig. S1. H&E staining of murine embryonic limb buds and hMSC sheets used for preparation of mesenchymal condensations.
    • Fig. S2. Fixation plate characterization.
    • Fig. S3. Bone accumulation and bridging rates.
    • Fig. S4. MicroCT ROI analysis demonstrating formation of cortical and trabecular bone compartments.
    • Fig. S5. Full histological analysis of endochondral bone formation at week 4.
    • Fig. S6. Full histological analysis of endochondral bone formation at week 12.
    • Fig. S7. Collagen organization.
    • Fig. S8. Histology of contrast agent–perfused sections.
    • Fig. S9. Best subsets analysis of mechanical testing data, models 2 to 5.
    • Fig. S10. Vasculature in defect periphery.
    • Fig. S11. mRNA expression of hMSCs in dynamically compressed hydrogels.
    • Legend for movie S1
    • References (7476)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Fixation plate actuation and bone regeneration.
    • Data file S1 (Microsoft Excel format). Primary data.

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