Dual-Phase Osteogenic and Vasculogenic Engineered Tissue for Bone Formation

Abstract

Minimally invasive, injectable bone tissue engineering therapies offer the potential to facilitate orthopedic repair procedures, including in indications where enhanced bone regeneration is needed for complete healing. In this study, we developed a dual-phase tissue construct consisting of osteogenic (Osteo) and vasculogenic (Vasculo) components. A modular tissue engineering approach was used to create collagen/fibrin/hydroxyapatite (COL/FIB/HA) hydrogel microbeads containing embedded human bone marrow-derived mesenchymal stem cells (bmMSC). These microbeads were predifferentiated toward the osteogenic lineage in vitro for 14 days, and they were then embedded within a COL/FIB vasculogenic phase containing a coculture of undifferentiated bmMSC and human umbilical vein endothelial cells (HUVEC). In vitro studies demonstrated homogenous dispersion of microbeads within the outer phase, with endothelial network formation around the microbeads over 14 days in the coculture conditions. Subcutaneous injection into immunodeficient mice was used to investigate the ability of dual-phase (Osteo+Vasculo) and control (Osteo, Vasculo, Blank) constructs to form neovasculature and ectopic bone. Laser Doppler imaging demonstrated blood perfusion through all constructs at 1, 4, and 8 weeks postimplantation. Histological quantification of total vessel density showed no significant differences between the conditions. Microcomputed tomography indicated significantly higher ectopic bone volume (BV) in the Osteo condition at 4 weeks. At 8 weeks both the Osteo and Blank groups exhibited higher BV compared to the Vasculo and dual Osteo+Vasculo groups. These data not only show that osteogenic microbeads can be used to induce ectopic bone formation, but also suggest an inhibitory effect on BV when undifferentiated bmMSC and HUVEC were included in dual-phase constructs. This work may lead to improved methods for engineering vascularized bone tissue, and to injectable therapies for the treatment of orthopedic pathologies in which bone regeneration is delayed or prevented.

Inosculation and perfusion of pre-vascularized tissue patches containing aligned human microvessels after myocardial infarction

“…6,12–18 The most impressive of these studies demonstrate that implantation of pre-cultured human microvessels in immune-compromised mice can result in inosculation with the host vasculature and perfusion of the human microvessels. 13,14,17 However, these studies were carried out in the healthy subcutaneous environment, and no study has implanted pre-cultured human microvessels in the especially challenging myocardial infarct environment.…”

Synergistic enhancement of ectopic bone formation by supplementation of freshly isolated marrow cells with purified MSC in collagen–chitosan hydrogel microbeads

“…It has been reported that undifferentiated MSC seeded on calcium phosphate bone grafts and implanted subcutaneously begin to disintegrate at day 3 and are completely absent by 14 days post-implantation (42). A similar study found that scaffolds seeded with undifferentiated MSC were filled with only fibro-vascular tissue and produced no new bone formation over 4 weeks in a subcutaneous implant (19), and our recent work has also suggested that undifferentiated MSC can inhibit bone formation (43). In contrast, the osteogenically differentiated culture-expanded MSC in our study showed robust ectopic bone formation.…”

“…The collagen–chitosan material was mildly osteogenic in the absence of cells, and served as a practical and osteoconductive carrier for the encapsulated cells, while also allowing facile pre-culture and minimally invasive delivery. The microbead format allowed pre-culture and minimally invasive delivery of the cell-biomaterial therapy, and had also been used to create multiphase tissues designed to potentiate tissue vascularization (43,47,48). This cell delivery system may have utility as an alternative to current bone grafting materials in applications where potent and rapid bone regeneration is needed.…”

“…Marrow-derived multipotent mesenchymal stem cells (MSC) are commonly investigated in the context of bone regeneration (1–3), due to their well-established self-renewal and osteoblastic differentiation potential. In addition, these cells exhibit important trophic and immunosuppressive activities that enhance the regenerative microenvironment (4). For bone regeneration applications, MSC are typically expanded in vitro to obtain large numbers of cells, but this process is time-consuming and is predicted to have high labor demand and facility costs for clinical-scale manufacturing (5).…”

Collagen/fibrin microbeads as a delivery system for Ag-doped bioactive glass and DPSCs for potential applications in dentistry

“…However, this surrounding insoluble matrix cannot provide biological cues to incorporated mammalian cells, as these cells do not have receptors for attachment to these polysaccharide materials. Because of this, other materials like fibrin, collagen, as well as blends of materials, have been used in order to enhance cell-matrix interactions with microbeads [14,[19][20][21].…”