Research ArticleBIOMATERIALS

An engineered cell-laden adhesive hydrogel promotes craniofacial bone tissue regeneration in rats

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Science Translational Medicine  11 Mar 2020:
Vol. 12, Issue 534, eaay6853
DOI: 10.1126/scitranslmed.aay6853
  • Fig. 1 Adhesive hydrogel synthesis and characterization.

    (A) Schematic illustration of chemical modification of alginate to make alginate-based adhesive hydrogel (AdhHG) that is photopolymerized either via visible light (eosin Y)– or UV (Irgacure 2959)–based photoinitiators. (B) Visualization of light-cured synthesized hydrogel and (C) its microstructure via scanning electron microscopy. Scale bar, 100 μm. (D) UV-vis and (E) H-NMR spectra of synthesized AdhHG. Full factorial investigation of methacrylation degree (0 to 22%) and degree of dopamine (DA) conjugation (0 to 4 mol%) on (F) swelling degree and (G) cumulative amount of release of sample protein [BSA–fluorescein isothiocyanate] after 48 hours (n = 3). (H) In vitro degradation based on mass loss of the hydrogels with and without presence of dopamine/methacrylate groups after incubation of hydrogels in either PBS or human saliva. The estimated half-life of different formulations is shown on the right (n = 3). Photographs illustrating hydrogel adhesiveness to (I) rat gingiva, (J) rat calvarial bone and periosteum, and (K) human tooth root surfaces. (L) Sequential images of tensile experiment on rat alveolar bone adhesion. (M) Stress-strain curve to identify adhesion strength and adhesion energy to porcine skin (n = 5). Sample preparation for adhesion test on porcine skin is shown in (N). The data are expressed as means ± SD. The results were statistically analyzed using one-way ANOVA with post hoc analysis. For all tests, ***P < 0.001 for differences between samples with different formulations. NS, not significant.

  • Fig. 2 In vitro cytocompatibility and in vivo biocompatibility of adhesive hydrogel.

    (A) In vitro biocompatibility of encapsulated GMSCs inside hydrogel beads and Live/Dead staining fluorescence images of GMSC-loaded alginate RGD, alginate-dopamine, and AdhHG hydrogels. Scale bars, 500 μm. (B) Quantitative Live/Dead results after 1 week of culturing in standard stem cell media. (P > 0.05; n = 5). (C) In vivo biocompatibility of designed hydrogels. Immunostaining of hydrogel boundaries 7 days after subcutaneous implantation in mice (* indicates hydrogel). DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 100 μm. (D) Evaluation of macrophages (CD68) and lymphocytes (CD3) infiltration surrounding the hydrogel implants (n = 5). (E) Whole-blood analysis of mice after implantation with various hydrogel formulations. Values were normalized to those for Alg-RGD. White blood cells: WBC (white blood cells), NE (neutrophils), LY (lymphocytes), MO (monocytes), EO (eosinophils), BA (basophils). Red blood cells: HCT (hematocrit), RBC (red blood cells), HB (hemoglobin), MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), RDW (red cell distribution width), RSD (reflex sympathetic dystrophy syndrome), RETIC (reticulocytes). Platelets: PLT (platelet count), MPV (mean platelet volume), PDW (platelet distribution width), and PCT (plateletcrit). (F) Comprehensive metabolic screening of mice after implantation with various hydrogel formulations. Values were normalized to those for Alg-RGD. Liver function assessment: ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), LDH (lactate dehydrogenase). Kidney function assessment: CREAT (creatinine), GLU (glucose). Electrolytes: CA (calcium), CO2 (carbon dioxide), MG (magnesium), and PHOS (potassium). (G) H&E staining analyzing degradation of the engineered adhesive hydrogel after 1 or 6 weeks of subcutaneous implantation in mice. CT, connective tissue; Alg, unresorbed adhesive alginate hydrogel. Scale bars, 200 μm.

  • Fig. 3 In vitro osteogenesis of stem cell aggregates encapsulated in adhesive hydrogel.

    Encapsulated cell aggregates in adhesive hydrogel promote osteogenic differentiation. (A) Schematic of the forced aggregation process to prepare GMSC and hydroxyapatite (HA) aggregates. (B) Viability (quantitative Live/Dead assay) of single cells and cell aggregates after 1 and 7 days of culture in control hydrogel (Alg-RGD; n = 5). Formation of cell GMSC aggregates (C) without and (D) with HAp MPs (cell:HAp 1:1) inside microwells immediately after seeding (left columns) and after 24 hours of culture (middle columns). Spheroids were removed from the wells and maintained in suspension culture (right columns). (E) qPCR demonstrating effects of different HAp MP:cell ratios on expression of osteogenic genes (n = 3). Alizarin red staining of single cells (F) and GMSC aggregates (G) encapsulated in AlgHG after 4 weeks of culturing in osteogenic media. Insets are xylenol orange staining under the same conditions. (H) Quantitative measurement of mineralization. Data are expressed as means ± SD. The results were statistically analyzed using oneway ANOVA with post hoc analysis. For all tests, *P < 0.05 and **P < 0.01 for differences between samples with different formulations.

  • Fig. 4 In vivo analyses of bone regeneration 8 weeks after subcutaneous implantation in immunocompromised mice.

    (A) Faxitron digital in vivo 2D radiographs of subcutaneously implanted hydrogels in the absence (upper panels) or presence of ca. 4 × 106 human GMSCs (middle panels) or an equal number of cells in aggregate form (±HA) (lower panels) per milliliter of Alg (alginate-RGD) ± 2 wt% HAp or AdhHG (±2 wt% HA). Boxed region is shown at higher magnification on the left in each column. (B) 3D reconstruction of micro-CT imaging of implanted hydrogels loaded with GMSCs, GMSC aggregates, or no cells. (C) Quantified relative mineralized density as normalized to mouse bone density and (D) bone volume (BV) fraction measurement derived from BV/total implanted volume (TV). (E) H&E staining of formulations of adhesive hydrogels with GMSCs, GMSC aggregates, or no cells 8 weeks after subcutaneous implantation in nude mice. (F) Quantified mineralized tissue from (E). The presented data are expressed as means ± SD (n = 5). The results were statistically analyzed using one-way ANOVA with post hoc analysis. For all tests, *P < 0.05, **P < 0.01, and ***P < 0.001 for differences between samples with different formulations.

  • Fig. 5 GMSC-mediated bone repair and regeneration in a rat model of peri-implantitis.

    (A) Photograph, schematic, and 3D micro-CT images of development of a rat model of A. actinomycetemcomitans–induced peri-implantitis. A. actinomycetemcomitans–coated titanium dental implants caused formation of peri-implantitis. Light-curable adhesive hydrogels with different formulations were injected into the defect, and healing was assessed at various time points afterward. (B) Survival of titanium dental implants was assessed over a period of 8 weeks (n = 4). Dashed white circles indicate the location of a lost implant. (C) 3D micro-CT images of animals treated with optimized formulation of adhesive or control hydrogels with or without cells at 8 weeks after implantation (n = 5). (D) Quantitative analysis of the amounts of bone fill at the peri-implant defect site 4 and 8 weeks after implantation. (E) Quantification of anti-inflammatory (IL-10) and inflammatory (TNF-α) cytokines at different time intervals (1, 2, 3, 4, and 5 weeks) after implantation (n = 4). The presented data are expressed as means ± SD. The results were statistically analyzed using one-way ANOVA with post hoc analysis. For all tests, *P < 0.05, **P < 0.01, and ***P < 0.001 for differences between samples with different formulations.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/534/eaay6853/DC1

    Fig. S1. Batch-to-batch variation of synthesized hydrogels (physical evaluation).

    Fig. S2. Batch-to-batch variation of synthesized hydrogels (cellular evaluation).

    Fig. S3. Shelf-life evaluation of synthesized hydrogels.

    Fig. S4. Adhesive strength of synthesized hydrogels compared with fibrin glue.

    Fig. S5. Osteogenic differentiation of GMSCs within adhesive hydrogels.

    Fig. S6. Rheological evaluation of adhesive hydrogel.

    Fig. S7. Osteogenic gene expression of GMSCs within adhesive hydrogels.

    Fig. S8. Growth factor gene expression of GMSCs within adhesive hydrogels.

    Fig. S9. Mineralization of single GMSCs and GMSC aggregates encapsulated in adhesive hydrogels.

    Fig. S10. In vivo bone formation using single GMSCs and GMSC aggregates encapsulated in adhesive hydrogels.

    Fig. S11. In vitro degradation of hydrogels in human saliva.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Batch-to-batch variation of synthesized hydrogels (physical evaluation).
    • Fig. S2. Batch-to-batch variation of synthesized hydrogels (cellular evaluation).
    • Fig. S3. Shelf-life evaluation of synthesized hydrogels.
    • Fig. S4. Adhesive strength of synthesized hydrogels compared with fibrin glue.
    • Fig. S5. Osteogenic differentiation of GMSCs within adhesive hydrogels.
    • Fig. S6. Rheological evaluation of adhesive hydrogel.
    • Fig. S7. Osteogenic gene expression of GMSCs within adhesive hydrogels.
    • Fig. S8. Growth factor gene expression of GMSCs within adhesive hydrogels.
    • Fig. S9. Mineralization of single GMSCs and GMSC aggregates encapsulated in adhesive hydrogels.
    • Fig. S10. In vivo bone formation using single GMSCs and GMSC aggregates encapsulated in adhesive hydrogels.
    • Fig. S11. In vitro degradation of hydrogels in human saliva.

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    Other Supplementary Material for this manuscript includes the following:

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