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. 2018 Mar 9;9(1):60.
doi: 10.1186/s13287-018-0790-8.

Polymer-mineral scaffold augments in vivo equine multipotent stromal cell osteogenesis

Wei Duan  1 , Cong Chen  2 , Masudul Haque  1 , Daniel Hayes  2 , Mandi J Lopez  3
Affiliations

Polymer-mineral scaffold augments in vivo equine multipotent stromal cell osteogenesis

Wei Duan et al. Stem Cell Res Ther. .

Abstract

Background: Use of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration.

Methods: This study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC) and adipose tissue-derived MSCs (ASC) were culture expanded to P3 and immunophenotyped with flow cytometry. They were then loaded by spinner flask on to scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-L-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Scaffolds with and without cells were maintained in static culture for up to 21 days or implanted subcutaneously in athymic mice that were radiographed every 3 weeks up to 9 weeks. In vitro cell viability and proliferation were determined. Explant composition (double-stranded (ds)DNA, collagen, sulfated glycosaminoglycan (sGAG), protein), equine and murine osteogenic target gene expression, microcomputed tomography (μCT) mineralization, and light microscopic structure were assessed.

Results: The ASC and BMSC number increased significantly in HT constructs between 7 and 21 days of culture, and BMSCs increased similarly in GT constructs. Radiographic opacity increased with time in GT-BMSC constructs. Extracellular matrix (ECM) components and dsDNA increased significantly in GT compared to HT constructs. Equine and murine osteogenic gene expression was highest in BMSC constructs with mineral-containing scaffolds. The HT constructs with either cell type had the highest mineral deposition based on μCT. Regardless of composition, scaffolds with cells had more ECM than those without, and osteoid was apparent in all BMSC constructs.

Conclusions: In this study, both exogenous and host MSCs appear to contribute to in vivo osteogenesis. Addition of mineral to polymer scaffolds enhances equine MSC osteogenesis over polymer alone, but pure mineral scaffold provides superior osteogenic support. These results emphasize the need for bioscaffolds that provide customized osteogenic direction of both exo- and endogenous MSCs for the best regenerative potential.

Keywords: Adipose; Bioreactor; Bone; Computed tomography; Microstructure; Murine.

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Conflict of interest statement

Ethics approval

All animal procedures were approved by the Louisiana State University Institutional Animal Care and Use Committee prior to study initiation (protocols #13-050 and 07-049).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Schematic of spinner flask bioreactor cell loading, scaffold division, and implantation
Fig. 2
Fig. 2
Immunophenotypes of P3 equine adipose-derived multipotent stromal cells (ASCs) and bone marrow-derived multipotent stromal cells (BMSCs) after culture expansion post-cryopreservation. The black lines represent labeled cells and the green lines represent autofluorescence
Fig. 3
Fig. 3
Fold-change in adipose-derived multipotent stromal cell (ASC; a) or bone marrow-derived multipotent stromal cell (BMSC; b) number after 7 or 21 days of static culture in stromal medium on scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Columns with distinct superscripts are significantly different between culture times within scaffold composition and those with different asterisk (*) numbers are significantly different among scaffold compositions within culture time (p < 0.05)
Fig. 4
Fig. 4
Radiographs of mice with carrier scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (GA) or PEG/PLLA/TCP/HA (GT) with no cells or equine adipose-derived multipotent stromal cells (ASCs) or bone marrow-derived multipotent  stromal cells (BMSCs) 0, 6, and 9 weeks after surgical implantation. White circles surround radiopaque implants
Fig. 5
Fig. 5
Three-dimensional explant reconstructions demonstrating models generated with high (left) and low (right) thresholds to distinguish between high contrast scaffold structure (left) versus low contrast newly deposited tissue (right). A single threshold was used for GA constructs due to the limited presence of high contrast material in the specimens. ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 6
Fig. 6
Percent porosity (a) and bone volume/total volume (BV/TV; b) (mean ± SEM) of equine ASC and BMSC constructs 9 weeks after subcutaneous implantation in a murine model. Columns with distinct superscripts are significantly different among cell tissue source within scaffolds and those with different asterisk (*) numbers are significantly different among scaffolds within cell tissue source (p < 0.05). ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 7
Fig. 7
The double-stranded DNA (dsDNA; a), hydroxyproline (collagen; b), sulfated glycosaminoglycan (sGAG; c) and protein (d) content (mean ± SEM) in equine ASC and BMSC constructs 9 weeks after subcutaneous implantation in a murine model. Columns with distinct superscripts are significantly different among cell types within scaffolds and those with different asterisk (*) numbers are significantly different among scaffolds within cell types (p < 0.05). ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 8
Fig. 8
Scanning electron photomicrographs of scaffolds before cell loading (preimplantation) and 9 weeks after implantation without (no cell) or combined with equine ASCs or BMSCs. Magnification = 3000×; scale bar = 20 μm. Yellow arrows show collagen fibrils; black arrows show solid (mineralizing) region. ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent  stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 9
Fig. 9
Energy dispersive x-ray microanalysis of explants before (preimplantation) cell loading or combined with equine ASCs or BMSCs 9 weeks after implantation. ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 10
Fig. 10
Equine alkaline phosphatase (ALP; a), osteocalcin (OCN; b), osteoprotegerin (OPG; c), and bone sialoprotein (BSP; d) levels (mean ± SEM) in equine MSC-scaffold constructs 9 weeks after implantation. Columns with distinct superscripts are significantly different between cell tissue source among scaffolds and those with different asterisk (*) numbers are significantly different among scaffolds between cell tissue source (p < 0.05). ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 11
Fig. 11
Murine alkaline phosphatase (ALP; a), osteocalcin (OCN; b), osteoprotegerin (OPG; c), and bone sialoprotein (BSP; d) levels (mean ± SEM) in equine MSC-scaffold constructs 9 weeks after implantation. Columns with distinct superscripts are significantly different between cell tissue source among scaffolds and those with different asterisk (*) numbers are significantly different among scaffolds between cell tissue source (p < 0.05). ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
Fig. 12
Fig. 12
Light photomicrographs of equine MSC-scaffold explants 9 weeks after surgery. Masson’s trichrome stain. Magnification = 10×; scale bar = 200 μm (a); magnification = 40×; scale bar = 50 μm (b). Black arrows show osteoid, yellow arrows show collagen fibers; gray arrows show proteinaceous ECM; and two-stripe black arrows show scaffold. ASC adipose-derived multipotent stromal cell, BMSC bone marrow-derived multipotent stromal cell, GA polyethylene glycol (PEG)/poly-l-lactic acid (PLLA), GT PEG/PLLA/tricalcium phosphate (TCP)/hydroxyapatite (HA), HT TCP/HA
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