A bioactive composite scaffold enhances osteochondral repair by using thermosensitive chitosan hydrogel and endothelial lineage cell-derived chondrogenic cell

Abstract

Articular cartilage regeneration is a major challenge in orthopedic medicine. Endothelial progenitor cells (EPCs) are a promising cell source for regenerative medicine applications. However, their roles and functions in cartilage regeneration are not well understood. Additionally, thermosensitive chitosan hydrogels have been widely used in tissue engineering, but further development of these hydrogels incorporating vascular lineage cells for cartilage repair is insufficient. Thus, this study aimed to characterize the ability of EPCs to undergo endothelial-mesenchymal stem cell transdifferentiation and chondrogenic differentiation and investigate the ability of chondrogenic EPC-seeded thermosensitive chitosan-graft-poly (N-isopropylacrylamide) (CEPC-CSPN) scaffolds to improve healing in a rabbit osteochondral defect (OCD) model. EPCs were isolated and endothelial-to-mesenchymal transition (EndMT) was induced by transforming growth factor-β1 (TGF-β1); these EPCs are subsequently termed transdifferentiated EPCs (tEPCs). The stem cell-like properties and chondrogenic potential of tEPCs were evaluated by a series of in vitro assays. Furthermore, the effect of CEPC-CSPN scaffolds on OCD repair was evaluated. Our in vitro results confirmed that treatment of EPC with TGF-β1 induced EndMT and the acquisition of stem cell-like properties, producing tEPCs. Upon inducing chondrogenic differentiation of tEPCs (CEPCs), the cells exhibited significantly enhanced chondrogenesis and chondrocyte surface markers after 25 days. The TGF-β1-induced differentiation of EPCs is mediated by both the TGF-β/Smad and extracellular signal-regulated kinase (Erk) pathways. The CEPC-CSPN scaffold reconstructed well-integrated translucent cartilage and repaired subchondral bone in vivo, exhibiting regenerative capacity. Collectively, our results suggest that the CEPC-CSPN scaffold induces OCD repair, representing a promising approach to articular cartilage regeneration.

Keywords: Chondrogenesis; Endothelial progenitor cells; Endothelial-to-mesenchymal transition; Osteochondral regeneration; Thermosensitive injectable hydrogels.

Graphical abstract

Fig. 1

EPCs derived from peripheral blood differentiate into multiple cell types under TGF-β1 stimulation. (a) Schematic of cell culture and differentiation. (b) Changes in the cultured EPCs: (i) PBMCs immediately after plating, (ii) PBMCs 2 days after seeding, (iii) PBMCs 7 days after seeding, and (iv) exponential growth of LEPCs 14 days after plating (10 × magnification; scale bar: 100 μm). (c) Changes in LEPC morphology after TGF-β1 stimulation (days 0 and 7): (i, iii) LEPCs or (ii, iv) TGF-β1-stimulated LEPCs (10 × magnification; scale bar: 100 μm). Abbreviations: EGM-2, endothelial growth medium-2; EPCs, endothelial progenitor cells; LEPCs, late EPCs; PBMCs, peripheral blood mononuclear cells; TGF-β1, transforming growth factor-β1.

Fig. 2

EndMT-derived tEPCs acquire an MSC-like phenotype. (a) Flow cytometry analysis of CD44, CD90, CD105 (mesenchymal stem cell markers), CD34 (hematopoietic and endothelial cell marker), and CD45 (leukocyte marker) expression in tEPCs. The empty areas show isotype control staining. The red-filled areas represent the expression of specific markers. (b) Representative immunofluorescence images of tEPC surface markers. tEPCs stain positive for MSC markers (CD44, CD90, and CD105) and negative for endothelial cell markers (CD31, eNOS, VE-cadherin, and vWF) (20 × magnification; scale bar: 50 μm). (c, d) CFU efficiency of EPCs and tEPCs, assessing self-renewal through the rate of colony formation in CFU assays. (c) Representative colonies of EPCs and tEPCs in 6-well plates (scale bar: 5 mm). (d) Columns illustrate the CFU efficiency. Values are reported as the mean ± standard deviation (SD) of six replicates. ***P < 0.001. (e) Multilineage differentiation potential of tEPCs induced to differentiate into (i) chondrogenic (10 × magnification; scale bar: 100 μm), (ii) osteogenic (10 × magnification; scale bar: 100 μm), or (iii) adipogenic (40 × magnification; scale bar: 10 μm) lineages. Abbreviations: CD, cluster of differentiation; CFU, colony-forming unit; EndMT, endothelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; MSC, mesenchymal stem cell; tEPC, transdifferentiated EPCs; VE-cadherin, vascular endothelial cadherin; vWF, von Willebrand factor.

Fig. 3

TGF-β1 induces tEPC activation and differentiation into chondrocyte-like cells that secrete cartilage-specific proteins. (a) Morphology of tEPCs after chondrogenic induction for 25 days: (i, iii) EPCs (control group) and (ii, iv) tEPCs after chondrogenic induction (CEPC group). EPCs and CEPCs were stained with Alcian Blue to assess the sGAG content (10 × magnification; scale bar: 100 μm). (b) sGAG synthesis in EPCs and CEPCs. (c) Intracellular collagen synthesis by EPCs and CEPCs. (d) mRNA expression of chondrogenic genes (ACAN/SOX9/Col II) and the fibrocartilage marker, Col I, in EPCs and CEPCs (day 25). Expression levels are expressed relative to those of the control group (defined as 1). Values are reported as the mean ± standard deviation (SD) of four replicates. *P < 0.05, **P < 0.01, and ***P < 0.001. (e) Flow cytometric confirmation of CD49c and CD151 expression on the surface of CEPCs, with up to 99 % of the CEPC population staining positive for CD49c (red area) and CD151 (red area) compared with isotype control CEPCs (white area). Abbreviations: ACAN, aggrecan; CD, cluster of differentiation; CEPCs, chondrogenic EPCs; Col I, collagen type I; Col II, collagen type II; EPCs, endothelial progenitor cells; sGAG, sulfated glycosaminoglycan; SOX9, sex-determining region Y-box 9; tEPCs, transdifferentiated EPCs; TGF-β1, transforming growth factor-β1.

Fig. 4

TGF-β signaling drives EndMT progression and regulates the chondrogenic differentiation of EPCs. CEPCs generated from EPCs were induced to undergo EndMT (via treatment with TGF-β1 for 7 days; tEPCs) and chondrogenic differentiation (for another 25 days). (a) Immunofluorescence (20 × magnification; scale bar: 50 μm) and (b) immunocytochemical (10 × magnification; scale bar: 10 μm) staining of (v–viii, xiii–xvi) EPCs and (i–iv, ix–xii) CEPCs in vitro. CEPCs stain positive for TGF-βRII, P-Smad2/3, Snail, P-Erk1/2, SOX9, and Col I/II (green and brown). DAPI counterstaining (blue). (c) Protein expression of EndMT signaling molecules (TGF-βRII, P-Smad2/3, Smad2/3, and Snail) in EPCs and CEPCs (day 25). (d) Protein expression of TGF-βRII, P-Smad2/3, Smad2/3, and Snail in EPCs and CEPCs after being normalized to β-actin (day 25). Values are reported as the mean ± standard deviation (SD) of three replicates. **P < 0.01 and ***P < 0.001. Abbreviations: CEPCs, chondrogenic EPCs; Col I, collagen type I; Col II, collagen type II; DAPI, 4′,6-diamidino-2-phenylindole; ECM, extracellular matrix; EndMT, endothelial-to-mesenchymal transition; EPCs, endothelial progenitor cells; Erk, extracellular signal-regulated kinase; P, phosphorylated; SOX9, sex-determining region Y-box 9; TGF-βRII, TGF-β type II receptor; tEPCs, transdifferentiated EPCs; TGF-β, transforming growth factor-β.

Fig. 5

Schematic illustration of the surgical procedure for implantation of a cellular scaffold in a full-thickness osteochondral injury within the weight-bearing area of the medial femoral condyle of a rabbit. MNC, mononuclear cell; EPCs, endothelial progenitor cells; tEPCs, transdifferentiated EPCs; CEPCs, chondrogenic EPCs; EndMT, endothelial-to-mesenchymal transition; CSPN, chitosan-graft-poly (N-isopropylacrylamide); EPC-CSPN, CSPN scaffold containing EPCs; CEPC-CSPN, CSPN scaffold containing CEPCs.

Fig. 6

Damaged cartilage can be effectively repaired by applying CEPC-based scaffolds to the affected area. (a) Representative photographs show the gross appearances of the scaffolds or cell scaffolds in cartilage defects at 4 and 12 weeks post-operation. Yellow circles represent the cartilage defect sites. Scale bar: 2 mm. (b) Quantitative gross appearance scores calculated at 4 and 12 weeks post-surgery. The reported values are the average of n = 6 ± standard deviation. *P < 0.05 and **P < 0.01. (c) Representative in vitro fluorescence imaging of DiI-labeled cells in the EPC-CSPN and CEPC-CSPN constructs in the cartilage defects at weeks 4 and 12 post-operation. (i) and (iii): Fluorescence images of EPCs in the EPC-CSPN constructs at the defect sites. (ii) and (iv): Fluorescence images of CEPCs in the CEPC-CSPN constructs at the defect sites. White circles represent cartilage defect sites, whereas the yellow areas represent DiI-labeled cells. (d) Fluorescence images obtained using fluorescence microscopy after paraffin sectioning. The EPC-CSPN and CEPC-CSPN constructs were implanted for 4 and 12 weeks. (i) and (iii): Fluorescence images of EPCs in the EPC-CSPN constructs at the defect sites. (ii) and (iv): Fluorescence images of CEPCs in the CEPC-CSPN constructs at the defect sites. The red areas represent DiI-labeled cells. Magnification: 4 × ; scale bar: 200 μm. Yellow arrows indicate the borders of defects. Abbreviations: C, cartilage layer; S, subchondral bone layer; EPCs, endothelial progenitor cells; CEPCs, chondrogenic EPCs; CSPN, chitosan-graft-poly (N-isopropylacrylamide); EPC-CSPN, CSPN scaffold containing EPCs; CEPC-CSPN, CSPN scaffold containing CEPCs.

Fig. 7

CSPN scaffolds require CEPCs to induce hyaline cartilage regeneration. Representative sections of defects filled with CSPN, EPC-CSPN, or CEPC-CSPN were stained using histochemical methods, including H&E (a), Masson's trichrome (b), and Safranin O/Fast Green staining (c). Sections were also subjected to immunostaining of collagen types II (d) and I (e). The area enclosed by the yellow box is shown enlarged underneath the main figure (a-iv-vi and a-x-xii). Black arrows indicate the borders of the defects. The original magnifications of the H&E images were 4 × (a-i–iii and a-vii–ix; scale bar: 200 μm) and 10 × (a-iv–vi and a-x–xiii; scale bar: 100 μm). The original magnifications of the Masson's trichrome (b-i–vii) and Safranin O/Fast Green (c-i–vii) staining images, immunostaining images for collagen types II (d-i–vii) and I (e-i–vii) were 10 × (Scale bar: 100 μm). Quantitative analysis of Safranin O (f) and collagen types II (g) and I (h) staining intensities, normalized relative to those in the CSPN group, which were defined as 1. The reported values are the average of n = 6 ± standard deviation. ***P < 0.001. Abbreviations: H&E, hematoxylin and eosin; EPCs, endothelial progenitor cells; CEPCs, chondrogenic EPCs; CSPN, chitosan-graft-poly (N-isopropylacrylamide); EPC-CSPN, CSPN scaffold containing EPCs; CEPC-CSPN, CSPN scaffold containing CEPCs.

Fig. 8

Using CEPCs induces an effective and rapid improvement in bone remodeling. (a) Two-dimensional (2D) micro-computed tomography images in the frontal plane for bone assessment, with red circles indicating the defect sites. (b) BV/TV. (c) Tb.Th. The reported values are the average of n = 6 ± standard deviation. *P < 0.05 and **P < 0.01. Abbreviations: BV/TV, the ratio of bone volume to tissue volume; Tb.Th, thickness of trabecular bone; CSPN, chitosan-graft-poly (N-isopropylacrylamide); EPC-CSPN, CSPN scaffold containing endothelial progenitor cells; CEPC-CSPN, CSPN scaffold containing chondrogenic endothelial progenitor cells.