Research ArticleTissue Engineering

Tissue-engineered autologous grafts for facial bone reconstruction

See allHide authors and affiliations

Science Translational Medicine  15 Jun 2016:
Vol. 8, Issue 343, pp. 343ra83
DOI: 10.1126/scitranslmed.aad5904
  • Fig. 1. Fabrication of engineered bone grafts.

    (A) Personalized bone tissue engineering process. Autologous MSCs (from fat aspirates) and CT images were obtained for each animal subject. The anatomical scaffold was fabricated from the bovine stifle bone that had been processed to remove any cellular material while preserving the tissue matrix. The cells were seeded into the scaffold and cultured in the specially designed perfusion bioreactor. After 3 weeks, the engineered RCU was implanted back into the pig for 6 months. (B) The bioreactor culture chamber consisted of five components designed to provide tightly controlled perfusion through the anatomical scaffold: anatomical inner chamber with bone scaffold, two halves of the polydimethylsiloxane (PDMS) (elastomer) block with incorporated channels, two manifolds, and an outer casing. The PDMS block was designed as an impression of the anatomical RCU structure and contained flow channels at both sides for the flow of culture medium in and out of the scaffold. The flow rate necessary for providing nutrient supply and hydrodynamic shear was defined by the design of parallel channels in the elastomer block (with the channel diameters and distribution dictated by the geometry of the graft) and the fluid-routing manifold. The channel diameters and spacing were specifically designed by flow simulation software for each pig to provide a desired interstitial flow velocity for a given shape and size of the anatomical RCU. (C) Flow simulation of the medium flow through the anatomically shaped scaffold reveals uniform flow velocity throughout the volume of the graft.

  • Fig. 2. Properties of engineered bone grafts.

    (A) DNA, ALP, and calcium (Ca) content over the course of 3 weeks of osteogenic induction. Significant increases in ALP and Ca deposition were observed. Data were quantified for each animal as means ± SD (n = 7 animals) using one-way analysis of variance (ANOVA) and Tukey’s comparison posttest. (B) ALP and von Kossa stains confirmed deposition of ALP protein and mineral during osteogenic induction. (C) DNA content quantifies the number of cells per scaffold at 3 days and 3 weeks after osteogenic induction in the scaffold-bioreactor system, as well as after transport (one additional day). Data are box plots (n = 7). P values were determined by one-way ANOVA with Tukey’s multiple comparison post hoc test. (D) Gene expression after 3 weeks of osteogenic induction of cells in the scaffold. Data are the fold difference (means ± SD) of gene expression normalized to the housekeeping gene (Gapdh) and then to the corresponding day 3 data (n = 7). P values were determined by t test with H0 = 1. (E) H&E staining and immunohistochemistry (type I collagen and BSP) of engineered bone grafts after seeding (3 days) and before implantation (3 weeks). Cells attached to the scaffold (black arrowheads), proliferated, filled the pore spaces, and deposited bone proteins (white arrowheads). The decellularized trabecular scaffold (S) was maintained throughout the cultivation period. Scale bars, 250 μm.

  • Fig. 3. Progression of bone regeneration and reestablishment of condyle height.

    (A) Lateral (left) and caudal (right) 3D views of the mandible after surgery. Images were taken at timed intervals for condylectomy controls (n = 1), animals implanted with acellular scaffolds (n = 2), and animals implanted with tissue-engineered bone (n = 2). Additional images are in fig. S2B. (B to D) Quantitative analysis of bone volume (B), bone volume fraction (C), and condyle height normalized to the original height (D). Data are individual animals with means ± SD (n = 6). ***P < 0.001; **P < 0.01; *P < 0.05, two-way ANOVA and Bonferroni post hoc tests.

  • Fig. 4. Morphology and structure of regenerated RCU.

    (A and B) Ramus regeneration was assessed by μCT (A) and Movat’s pentachrome staining for bone and soft tissues (B). Scale bars, 1 cm. (C and D) Condyle regeneration was assessed using μCT 3D reconstruction (C) and Movat’s pentachrome staining (D) at low magnification (top; 1-cm scale) and high magnification (bottom; 2-mm scale) of the condylectomy site. The dashed circumferences indicate the remaining graft regions, with the red trabecular structure representing the remaining scaffold material. Images are representative of n = 2 (condylectomy control), n = 4 (acellular scaffold), and n = 4 (tissue-engineered bone) animals.

  • Fig. 5. Graft-host bone integration.

    (A) μCT 3D reconstruction of the graft-host interface and Movat’s pentachrome staining were used to assess integration of the implanted graft with the host bone. For acellular grafts, the mineralized host bone (hb) and the graft structures (g) were separated by soft fibrous tissue (f). In contrast, host bone extended into the tissue-engineered bone graft. In the proximity of the new bone, osteoclastic resorption (white arrowheads) was detected on the implanted scaffold with the lining of osteoblasts (black arrowheads), indicating active ossification. Scale bars, 1 mm (4×) and 100 μm (40×). (B and C) Three-point bending mechanical test for peak and equilibrium flexural moduli (B) and the peak and equilibrium forces (C). Data are means ± SD (n = 4 from two animals for condylectomy; n = 4 from two animals for 3-month data; n = 8 from four animals for 6-month data). ***P < 0.001; **P < 0.01; *P < 0.05, two-way ANOVA and Bonferroni post hoc tests.

  • Fig. 6. Deposition of type I collagen and BSP in the newly formed bone.

    The regenerating new bone (nb) within the tissue-engineered bone graft is shown at low and high magnification. Localized areas of type I collagen and BSP at the graft-host bone interface were precursors for bone formation. New bone contained osteocytes (black arrowheads) within their lacunae. Negative immunohistochemistry confirmed the specificity of the staining. Images are representative of n = 4 animals. Scale bars, 400 μm (low) and 100 μm (high).

  • Fig. 7. Graft resorption and vascularization.

    (A) Movat’s pentachrome stain of the central region within the scaffolds is shown 3 months after implantation. Scaffold resorption is indicated by white arrows. Vasculature is indicated by black arrows. Scale bars, 1 mm and 250 μm. (B and C) Quantification of microstructure properties: percentage of scaffold area (B) and percentage of the resorbing area (C). (D) H&E showed structural differences between the newly formed vasculature and the surrounding tissue. Immunohistochemistry for CD31 confirmed the presence of endothelial cell lining in newly formed vessels in both tissue-engineered bone and acellular scaffold group. Negative control confirmed the specificity of CD31 antibody. The CD31 immunohistochemistry was used to quantify vascular number per area. Scale bar, 50 μm. (E) The number of blood vessels per area was quantified from CD31 immunohistochemical images. (B, C, and E) Data are box plots for n = 56 images for the acellular scaffold group and n = 90 images for the tissue-engineered bone group.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/343/343ra83/DC1

    Methods

    Fig. S1. Structural comparison of tissue-engineered graft and native explant.

    Fig. S2. Regeneration of RCU.

    Fig. S3. High-resolution Movat’s pentachrome stain of the ramus in the condylectomy group at 6 months.

    Fig. S4. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.

    Fig. S5. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bone group.

    Fig. S6. Condyle regeneration (posterior view and histologies).

    Fig. S7. High-resolution Movat’s pentachrome stain of the condyle in the condylectomy group at 6 months.

    Fig. S8. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.

    Fig. S9. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bone group.

    Fig. S10. Ossification patterns in the condyle after tissue-engineered bone regeneration.

    Fig. S11. Experimental setup for the three-point bending mechanical testing.

    Table S1. Porcine real-time polymerase chain reaction (RT-PCR) primers.

    Movie S1. Assembly of bioreactor.

    Movie S2. Finite element modeling of oxygen concentration.

  • Supplementary Material for:

    Tissue-engineered autologous grafts for facial bone reconstruction

    Sarindr Bhumiratana, Jonathan C. Bernhard, David M. Alfi, Keith Yeager, Ryan E. Eton, Jonathan Bova, Forum Shah, Jeffrey M. Gimble, Mandi J. Lopez, Sidney B. Eisig, Gordana Vunjak-Novakovic*

    *Corresponding author. Email: gv2131{at}columbia.edu

    Published 15 June 2016, Sci. Transl. Med. 8, 343ra83 (2016)
    DOI: 10.1126/scitranslmed.aad5904

    This PDF file includes:

    • Methods
    • Fig. S1. Structural comparison of tissue-engineered graft and native explant.
    • Fig. S2. Regeneration of RCU.
    • Fig. S3. High-resolution Movat’s pentachrome stain of the ramus in the condylectomy group at 6 months.
    • Fig. S4. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.
    • Fig. S5. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bone group.
    • Fig. S6. Condyle regeneration (posterior view and histologies).
    • Fig. S7. High-resolution Movat’s pentachrome stain of the condyle in the condylectomy group at 6 months.
    • Fig. S8. High-resolution Movat’s pentachrome stain of the ramus in the acellular scaffold group.
    • Fig. S9. High-resolution Movat’s pentachrome stain of the ramus in the tissue-engineered bone group.
    • Fig. S10. Ossification patterns in the condyle after tissue-engineered bone regeneration.
    • Fig. S11. Experimental setup for the three-point bending mechanical testing.
    • Table S1. Porcine real-time polymerase chain reaction (RT-PCR) primers.
    • Legends for Movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Assembly of bioreactor.
    • Movie S2 (.mp4 format). Finite element modeling of oxygen concentration.

    [Download Movies S1 and S2]

Stay Connected to Science Translational Medicine

Navigate This Article