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, 19 (13-14), 1641-53

Glycosaminoglycan Mimetic Associated to Human Mesenchymal Stem Cell-Based Scaffolds Inhibit Ectopic Bone Formation, but Induce Angiogenesis in Vivo

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Glycosaminoglycan Mimetic Associated to Human Mesenchymal Stem Cell-Based Scaffolds Inhibit Ectopic Bone Formation, but Induce Angiogenesis in Vivo

Guilhem Frescaline et al. Tissue Eng Part A.

Abstract

Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR4120] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR4120] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro. Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.

Figures

FIG. 1.
FIG. 1.
The glycosaminoglycan (GAG) mimetic [OTR4120] potentiates the human mesenchymal stem cell (hMSC) in vitro proliferation and migration properties. (A) hMSC proliferation was quantified using MTT assay after 5 days of treatment without (CT) or with increasing doses (ng/mL) of the GAG mimetic [OTR4120] solubilized into culture medium and compared to 100 ng/mL of dextran (T40), natural heparan sulfates (HS) and Heparin (Hep). (B) The migration capacity of hMSC toward soluble [OTR4120] was analyzed in the Boyden chambers assays for 12 h. Controls were performed in absence of fetal calf serum (FCS) as negative CT, and with 20% FCS and 5 ng/mL fibroblast growth factor-2 (FGF-2) as positive CT. Means±standard deviation (SD) were obtained from triplicate per condition and per experiment, and from three independent experiments (*p<0.05; **p<0.01; ***p<0.001).
FIG. 2.
FIG. 2.
The GAG mimetic [OTR4120] potentiates the hMSC in vitro commitment to the osteogenic lineage. hMSC were expanded in a control (culture medium [CM]) or osteogenic (OS) medium supplemented or not with [OTR4120] during 21 days. (A) Alizarin Red staining of the calcium deposits. Scale bar: 1 mm. (B) Flow cytometry analysis of alkaline phosphatase (ALP) expression. (C, D) Quantitative real-time polymerase chain reaction analysis of the expression of bone morphogenic protein-2 (BMP-2), osteocalcin (OC), and osteopontin (OP) after 7 days (C) and 14 days (D) of treatment. Expression levels are reported to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and presented as 2−ΔCt. Values are means±SD of triplicate per condition and per experiment, from three independent experiments (n=9; *p<0.05; **p<0.01). Color images available online at www.liebertpub.com/tea
FIG. 3.
FIG. 3.
The GAG mimetic [OTR4120] functionalizes tricalcium phosphate/hydroxyapatite (TCP/HA) bone substitutes. Scaffolds were engrafted with 4 or 20 μg of [OTR4120] per cm2 of the TCP/HA-developed surface, TCP/HA/OTR4120 [A], and [B] scaffolds, respectively, or with phosphate-buffered saline 1×(negative control), and colonized by hMSC before in vivo implantation. Four weeks later, the scaffolds were harvested and treated enzymatically to eliminate proteins and endogenous glycans (GAG mimetics are resistant to these treatments). (A) The output of [OTR4120] amount (ng/cm2) covalently engrafted and quantified by the dimethyl methylene blue (DMMB) assay at day 0 before implantation (D0 black plots) and 30 days after in vivo implantation (D30 white plots) (n=6; ***p<0.001). (B) Colonization efficiency of scaffolds by hMSC (means of cell number±SD) remaining into the scaffolds after 3 h of in vitro incubation before in vivo implantation (D0) and quantified with the fluorescent DNA probe Picogreen according to a standard curve of cells (n=8; **p<0.01; ***p<0.001). (C–H) In vivo localization and stability of [OTR4120]FITC engrafted on bone substitutes (F, G, H) compared to the control scaffolds without a GAG mimetic (C, D, E). Scaffolds were treated for DMMB assay (C, F) and embedded in methyl methacrylate for analysis by phase-contrast microscopy (D, G) and fluorescence microscopy (E, H). Red dotted line indicates outline of the scaffold. Ma, macropores; Mi, micropore; Ic, interconnection; Sc, scaffold; Mm, methyl methacrylate; Bo, border. Black scale bar: 1000 μm, white scale bar: 500 μm. Color images available online at www.liebertpub.com/tea
FIG. 4.
FIG. 4.
The GAG mimetic [OTR4120] immobilized on the TCP/HA substitutes inhibits bone formation induced by hMSC in the ectopic site. Controls and/or functionalized and/or cellularized scaffolds were implanted on mice and harvested after 8 weeks for histological study. (A) Masson's trichrome stain revealed the collagenic fibrillar tissue (arrow). (B) ALP activity staining showed violet areas with osteoblastic precursor cells (stars). (C) Toluidine blue staining indicated the host inflammatory response (arrow). (D) Tartrate-resistant acid phosphatase (TRAP) staining highlighted the red foci of osteoclastic cells (stars). Bn, bone nodule; Sc, scaffold; Bo: border. Scale bar: 200 μm. Pictures are representative of n=12 samples (2 substitutes per mice×2 mice×3 independent experiments). Semiquantitative analysis of this histological study was reported in Table 1. Color images available online at www.liebertpub.com/tea
FIG. 5.
FIG. 5.
The GAG mimetic [OTR4120] immobilized on the TCP/HA substitutes potentiates the in vivo vascularization of the devices. (A) Macroscopic views of the capillary network surrounding each series of constructs. White bar: 5 mm. (B, C) Automatic computerized vessel tree segmentation decomposed the capillary areas in a sum of objects discriminating intermediary (segments) and terminal (branches) degrees of ramification before (B) and after (C) modelization. Black scale bar: 2 mm. (D) Automatic computerized quantification of the vascular element number. Data are mean values±SD from n=12 samples (4 cardinal areas per scaffold×2 scaffolds per mice×2 mice per conditions×2 independent experiments; *p<0.05; **p<0.01). Color images available online at www.liebertpub.com/tea

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