Generation of a Bone Organ by Human Adipose-Derived Stromal Cells Through Endochondral Ossification

Abstract

: Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. Unlike bone marrow-derived stromal cells (also known as bone marrow-derived mesenchymal stromal/stem cells), adipose-derived stromal cells (ASC) have so far failed to form a bone organ by ECO. The goal of the present study was to assess whether priming human ASC to a defined stage of chondrogenesis in vitro allows their autonomous ECO upon ectopic implantation. ASC were cultured either as micromass pellets or into collagen sponges in chondrogenic medium containing transforming growth factor-β3 and bone morphogenetic protein-6 for 4 weeks (early hypertrophic templates) or for two additional weeks in medium supplemented with β-glycerophosphate, l-thyroxin, and interleukin1-β to induce hypertrophic maturation (late hypertrophic templates). Constructs were implanted in vivo and analyzed after 8 weeks. In vitro, ASC deposited cartilaginous matrix positive for glycosaminoglycans, type II collagen, and Indian hedgehog. Hypertrophic maturation induced upregulation of type X collagen, bone sialoprotein, and matrix metalloproteinase13 (MMP13). In vivo, both early and late hypertrophic templates underwent cartilage remodeling, as assessed by MMP13- and tartrate-resistant acid phosphatase-positive staining, and developed bone ossicles, including bone marrow elements, although to variable degrees of efficiency. In situ hybridization for human-specific sequences and staining with a human specific anti-CD146 antibody demonstrated the direct contribution of ASC to bone and stromal tissue formation. In conclusion, despite their debated skeletal progenitor nature, human ASC can generate bone organs through ECO when suitably primed in vitro.

Significance: Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. This study demonstrated that expanded, human adult adipose-derived stromal cells can generate ectopic bone through ECO, as previously reported for bone marrow stromal cells. This system can be used as a model in a variety of settings for mimicking ECO during development, physiology, or pathology (e.g., to investigate the role of BMPs, their receptors, and signaling pathways). The findings have also translational relevance in the field of bone regeneration, which, despite several advances in the domains of materials and surgical techniques, still faces various limitations before being introduced in the routine clinical practice.

Keywords: Adipose-derived stromal cells; Bone organ; Differentiation; Endochondral ossification; Tissue engineering.

Figure 1.

In vitro generation of cartilaginous tissues. Shown are representative fields of Safranin-O and Toluidine Blue staining for glycosaminoglycans (GAG) of engineered pellets (A, C) and collagen-based scaffolds (B, D) after 4 weeks of in vitro culture in chondro-inductive medium. Scale bar = 50 µm.

Figure 2.

In vitro maturation of cartilaginous tissues. (A): Safranin-O staining and immunostaining for Col II, Col X, MMP13, and BSP of early (4 weeks, left column) and late (4 + 2 weeks) hypertrophic scaffold-based samples (right column). (B): Quantification (mean ± SD) of mRNA expression of chondrogenic and early and late hypertrophic markers in early (black bars) and late (white bars) hypertrophic scaffolds. ∗∗∗, p < .001 (two-way ANOVA with Bonferroni post-tests, n = 4 values per group). Scale bar = 100 µm. Abbreviations: BMP4, bone morphogenetic protein-4; BMP7, bone morphogenetic protein-7; BSP, bone sialoprotein; Col II, collagen type II; Col X, collagen type X; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP13, matrix metalloproteinase13; runx 2, runt-related transcription factor 2; Saf-O, Safranin-O.

Figure 3.

In vitro activation of the IHH signaling pathway. Shown is in situ hybridization for IHH mRNA in early (4 weeks [A]) and late (4 + 2 weeks [B]) hypertrophic scaffold-based samples. (C): Quantification of mRNA expression (mean ± SD) for IHH, its receptor PTCH1, and GLI1 in early (white bar) and late (black bar) hypertrophic constructs. Scale bar = 50 µm. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLI1, glioma-associated oncogene homolog-1; IHH, Indian hedgehog; PTCH1, patched homolog-1; SHH, sonic hedgehog homolog.

Figure 4.

Development of the cartilaginous tissues following ectopic in vivo implantation. Shown is characterization of early (4 weeks [A]) and late (4 + 2 weeks [B]) hypertrophic scaffold-based constructs after 8 weeks of in vivo implantation by histochemical (H&E and Masson’s trichrome staining), histological, and immunohistochemical analysis. (C): Tomographic reconstruction of early and late hypertrophic scaffold-based constructs after 8 weeks of implantation. (D): Quantification of mineralized tissue volume in early (black bar) and late (white bar) hypertrophic scaffold-based constructs. ∗, p < .05 (two-way ANOVA with Bonferroni post-tests, n = 3 values per group). Scale bar = 100 µm. Abbreviations: ANOVA, analysis of variance; BSP, bone sialoprotein; BV/TV, bone volume/total volume; Col II, collagen type II; Col X, collagen type X; H&E, hematoxylin and eosin; Saf-O, Safranin-O.

Figure 5.

In vivo remodeling and vascularization. Immunohistochemical staining for MMP13 (top), Masson Trichrome staining for vascularization of the bone marrow cavity (white arrows, middle), and TRAP staining for osteoclasts/chondroclasts in early (4 weeks, bottom left) and late hypertrophic scaffold-based constructs (4 + 2 weeks, bottom right) are shown. Scale bar = 50 µm. Abbreviations: MMP13, matrix metalloproteinase13; TRAP, tartrate-resistant acid phosphatase.

Figure 6.

Human cells contribute to the formation of bone tissue and bone marrow stroma. Shown is in situ hybridization for human specific Alu sequences (blue staining, white arrows) in early (4 weeks [A, B]) and late (4 + 2 weeks [C, D]) hypertrophic scaffold-based constructs. (E, F): Human origin CD146+ cells (black arrows) in the marrow stroma. Scale bar = 50 µm. Abbreviations: B, bone; BM, bone marrow.