Directed Regeneration of Osteochondral Tissue by Hierarchical Assembly of Spatially Organized Composite Spheroids

Abstract

The use of engineered scaffolds or stem cells is investigated widely in the repair of injured musculoskeletal tissue. However, the combined regeneration of hierarchical osteochondral tissue remains a challenge due to delamination between cartilage and subchondral bone or difficulty in spatial control over differentiation of transplanted stem cells. Here, two types of composite spheroids are prepared using adipose-derived stem cells (hADSCs) and nanofibers coated with either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis, respectively. Each type of spheroid is then cultured within a 3D-printed microchamber in a spatially arranged manner to recapitulate the bilayer structure of osteochondral tissue. The presence of inductive factors regionally modulates in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct without dedifferentiation. Furthermore, hADSCs from each spheroid proliferate and sprout and successfully connect the two layers mimicking the osteochondral interface without apertures. In vivo transplantation of the biphasic construct onto a femoral trochlear groove defect in rabbit knee joint results in 21.2 ± 2.8% subchondral bone volume/total volume and a cartilage score of 25.0 ± 3.7. The present approach can be an effective therapeutic platform to engineer complex tissue.

Keywords: 3D printing; composite spheroid; microchamber; osteochondral tissue regeneration; tissue engineering.

Scheme 1

Schematic illustration of the formation of chondrogenic or osteogenic composite spheroids and the fabrication of biphasic construct positions with the spheroids for transplantation. Growth factors were immobilized on the fragmented fibers, which were hybridized with stem cells to form each type of spheroid. The spheroids were positioned hierarchically on each well of a 3D‐printed microchamber to form biphasic layers. The biphasic construct was transplanted onto an osteochondral defect for in vivo analysis.

Figure 1

Preparation of osteogenic or chondrogenic composite spheroids. a) Phase‐contrast images of PS, BS, and TS cultured for 21 d in vitro (white arrow: cell and fiber debris, scale bar = 250 µm). b) Size change of the spheroids over 21 d (n = 6). c) Relative DNA contents of the spheroids after 21 d compared with those of day 1 (n = 6). d) Osteogenic gene expression of hADSCs from PS, PS/B, and BS (n = 3) and e) the amount of deposited calcium ions from each spheroid after 21 d (n = 5). f) Chondrogenic gene expression of hADSCs from PS, PS/T, and TS (n = 3) and g) the amount of GAGs from each spheroid after 21 d (n = 5). h) Alizarin red S staining of the cross‐sectioned spheroids of PS, PS/B, and BS and i) alcian blue staining of the cross‐sectioned spheroids of PS, PS/T, and TS (scale bar = 200 µm). All statistical analyses were performed by one‐way analysis of variance (ANOVA). * = p < 0.05 and *** = p < 0.001.

Figure 2

Preparation of 3D‐printed microchambers and positioning of spheroids. a) Optical images of chambers and the number of amine groups from the microchambers depending on PD coating time (n = 4). b) Optical images of each microchamber with or without PD coating after dipping the chambers into media and loading the spheroids (scale bar = 4 mm). c) DNA content of the cells in each PS, BS, and TS loaded within chamber at days 1 and 21 (n = 4). d) Phase‐contrast images of a well organized with each PS, BS, and TS at day 1 (scale bar = 250 µm), and H&E staining and live and dead staining of each group after 21 d (scale bar = 200 µm). e) SEM images of the groups from the top view (scale bar = 500 µm), the portions with red boxes were enlarged in high magnification (scale bar = 100 µm), and their vertically cross‐sectioned images (scale bar = 500 µm). All statistical analyses were performed by one‐way analysis of variance (ANOVA). * = p < 0.05 and *** = p < 0.001.

Figure 3

In vitro differentiation of hADSCs from spheroids within the chambers. For osteogenic differentiation, a) alizarin red S and b) Col1a IHC staining of horizontally cross‐sectioned chambers carrying PS or BS (dotted lines: borderline of chamber well, scale bar = 200 µm). c) Deposited calcium contents and d) Col1a‐positive cells from each PS and BS loaded chamber (n = 6). e) Osteogenic gene expression of cells from the groups (n = 3). For chondrogenic differentiation, f) alcian blue and b) Col2a IHC staining of horizontally cross‐sectioned chambers carrying PS or TS (dotted lines: borderline of chamber well, scale bar = 200 µm). c) Amounts of GAGs and d) Col2a‐positive cells from each PS and TS positioned chamber (n = 6). e) Chondrogenic gene expression of the cells from the groups (n = 3). The statistical analyses for PCR investigations were performed by one‐way analysis of variance (ANOVA), and the others were by Student's t‐test. * = p < 0.05 and *** = p < 0.001.

Figure 4

In vitro integration of BS and TS organized microchambers to form a biphasic structure. a) Schematic illustration of the process to integrate the chambers carrying BS or TS, and the illustration of spheroids positioned inside an integrated well. SEM images of b) vertically cross‐sectioned (scale bar = 250 µm) and c) outside of BS/TS (scale bar = 500 µm) (green: cells; black dotted line: borderline between chambers). d) Phase‐contrast image of BS/TS from the top view (scale bar = 500 µm). e) Optical image of BS/TS being held out of culturing medium by forceps (white dotted line: borderline between chambers, scale bar = 4 mm).

Figure 5

In vitro differentiation of stem cells within biphasic construct. a) Schematic illustration of the process in a vertical cross section of BS/TS. b) Alizarin red S and c) alcian blue staining of vertically cross‐sectioned BS/TS (dotted line: borderline between BS and TS loaded microchambers, scale bar = 200 µm). d) Col1a IHC staining of vertically sectioned BS/TS (scale bar = 200 µm) and e) the ratio of Col1a‐positive cells in the red boxes (n = 6), and f) Col2a IHC staining of vertically cross‐sectioned BS/TS (scale bar = 200 µm) and g) the ratio of Col2a‐positive cells in the red boxes (n = 6) (Area 1: cells spread from BS, Area 2: cells near the borderline, Area 3: cells spread from TS). h) Schematic illustration of a horizontal cross section of BS/TS. i) OPN IHC staining of horizontally cross‐sectioned BS/TS and j) the ratio of OPN‐positive cells (n = 4), and k) SOX9 IHC staining of horizontally sectioned BS/TS and i) the ratio of SOX9‐positive cells (n = 4) (dotted line: a chamber well). The statistical analyses for the ratio of Col1a and Col2a positive cells were performed by one‐way analysis of variance (ANOVA), and the others were by Student's t‐test. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Figure 6

In vivo analysis after transplantation of spheroid‐laden biphasic construct. a) Optical images of harvested osteochondral tissues from Chamber, PS, and BS/TS groups (scale bar = 4 mm). b) X‐ray images (red boxes indicate defect area) and c) 3D images from μCT analysis of the Defect, Chamber, PS, and BS/TS groups (region of interest, ROI, indicating defect area is colored in orange, scale bar = 4 mm). d) BV/TV values from μCT analysis of the groups (n = 7), and the statistical analysis was performed by one‐way analysis of variance (ANOVA) (* = p < 0.05 and *** = p < 0.001). H&E staining of e) Defect, f) Chamber, g) PS, and h) BS/TS groups (scale bar = 1 mm) (the portions with “*” are in high magnification (scale bar = 100 µm); dotted line, defect area; CT, connective tissue; CB, compact bone; IB, immature bone; P, PCL chamber; FC, fibrocartilage; NB, new trabecular bone; NC, new articular cartilage).

Figure 7

Histological analysis and scoring of rabbit tissues. a) Safranin O and b) Masson's trichrome staining of harvested osteochondral tissues of the Defect, Chamber, PS, and BS/TS groups. Histological scoring for c) subchondral bone or d) cartilage morphology of the regenerated tissues from each group and e) the total scores for each parameter (n = 5), and the statistical analysis was performed by one‐way analysis of variance (ANOVA) (*** = p < 0.001).