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Visualizing osteogenesis in vivo within a cell-scaffold construct for bone tissue engineering using two-photon microscopy.

ABSTRACT: Tissue-engineering therapies have shown early success in the clinic, however, the cell-biomaterial interactions that result in successful outcomes are not yet well understood and are difficult to observe. Here we describe a method for visualizing bone formation within a tissue-engineered construct in vivo, at a single-cell resolution, and in situ in three dimensions using two-photon microscopy. First, two-photon microscopy and histological perspectives were spatially linked using fluorescent reporters for cells in the skeletal lineage. In the process, the tissue microenvironment that precedes a repair-focused study was described. The distribution and organization of type I collagen in the calvarial microenvironment was also described using its second harmonic signal. Second, this platform was used to observe in vivo, for the first time, host cells, donor cells, scaffold, and new bone formation within cell-seeded constructs in a bone defect. We examined constructs during bone repair 4 and 6 weeks after implantation. New bone formed on scaffolds, primarily by donor cells. Host cells formed a new periosteal layer that covered the implant. Scaffold resorption appeared to be site specific, where areas near the top were removed and deeper areas were completely embedded in new mineral. Visualizing the in vivo progression of the cell and scaffold microenvironment will contribute to our understanding of tissue-engineered regeneration and should lead to the development of more streamlined and therapeutically powerful approaches.

SUBMITTER: Villa MM

PROVIDER: S-EPMC3793663 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Visualizing osteogenesis in vivo within a cell-scaffold construct for bone tissue engineering using two-photon microscopy.

Villa Max M MM Wang Liping L Huang Jianping J Rowe David W DW Wei Mei M

Tissue engineering. Part C, Methods 20130608 11

Tissue-engineering therapies have shown early success in the clinic, however, the cell-biomaterial interactions that result in successful outcomes are not yet well understood and are difficult to observe. Here we describe a method for visualizing bone formation within a tissue-engineered construct in vivo, at a single-cell resolution, and in situ in three dimensions using two-photon microscopy. First, two-photon microscopy and histological perspectives were spatially linked using fluorescent rep ...[more]

PMID: 23641794

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Project description: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 (?CT) 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 ?CT. 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.

Dataset Information

Visualizing osteogenesis in vivo within a cell-scaffold construct for bone tissue engineering using two-photon microscopy.

Publications

Visualizing osteogenesis in vivo within a cell-scaffold construct for bone tissue engineering using two-photon microscopy.

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