An endochondral ossification approach to early stage bone repair: Use of tissue-engineered hypertrophic cartilage constructs as primordial templates for weight-bearing bone repair.

Mimicking endochondral ossification to engineer constructs offers a novel solution to overcoming the problems associated with poor vascularisation in bone repair. This can be achieved by harnessing the angiogenic potency of hypertrophic cartilage. In this study, we demonstrate that tissue-engineered hypertrophically primed cartilage constructs can be developed from collagen-based scaffolds cultured with mesenchymal stem cells. These constructs were subsequently implanted into femoral defects in rats. It was evident that the constructs could support enhanced early stage healing at 4 weeks of these weight-bearing femoral bone defects compared to untreated defects. This study demonstrates the value of combining knowledge of development biology and tissue engineering in a developmental engineering inspired approach to tissue repair.

The pre-vascularisation of a collagen-chondroitin sulphate scaffold using human amniotic fluid-derived stem cells to enhance and stabilise endothelial cell-mediated vessel formation.

A major problem in tissue engineering (TE) is graft failure in vivo due to core degradation in in vitro engineered constructs designed to regenerate thick tissues such as bone. The integration of constructs post-implantation relies on the rapid formation of functional vasculature. A recent approach to overcome core degradation focuses on the creation of cell-based, pre-engineered vasculature formed within the TE construct in vitro, prior to implantation in vivo. The primary objective of this study was to investigate whether an amniotic fluid-derived stem cell (AFSC)-human umbilical vein endothelial cell (HUVEC) co-culture could be used to engineer in vitro vasculature in a collagen chondroitin sulphate (CCS) scaffold. The secondary objective was to investigate whether hypoxic conditions (2% O2) could enhance microcapillary-like structure formation by this co-culture. The results of this study demonstrate, for the first time, that the AFSC-HUVEC co-culture was capable of pre-vascularising CCS scaffolds within 7 days and that the AFSCs are capable of behaving as pericytes while interacting with HUVECS to form microcapillary-like structures. However, this microcapillary-like structure formation was reduced in hypoxic conditions. qRT-PCR analysis indicated that an upregulation of VEGFR1 and accompanying decrease of VEGFR2 gene expression may be responsible for the poor response of these microcapillary-like structures to hypoxic conditions. Overall, however, these results demonstrate the potential of this newly developed co-culture system for the formation of pre-engineered vasculature within TE constructs. STATEMENT OF SIGNIFICANCE: This article describes the development of an amniotic fluid-derived stem cell (AFSC)-human umbilical vein endothelial cell (HUVEC) co-culture for use in engineering in vitro vasculature in a collagen chondroitin sulphate (CCS) scaffold. The article also describes the effect of hypoxic conditions on the networks of microcapillary-like structures formed by this co-culture. The AFSC-HUVEC co-culture was capable of pre-vascularising CCS scaffolds within 7 days. However, microcapillary-like structure formation was reduced in hypoxic conditions. Overall, these results demonstrate the potential of this newly developed co-culture system for the formation of pre-engineered vasculature within TE constructs. The proangiogenic nature of this co-culture has the potential to both enhance bone regeneration while also overcoming the problem of inadequate vascularisation of grafts commonly seen in the field of tissue engineering.

Investigating the effect of hypoxic culture on the endothelial differentiation of human amniotic fluid-derived stem cells.

Amniotic fluid-derived stem cells (AFSCs) are a unique stem cell source that may have great potential for use in tissue engineering (TE) due to their pluripotentiality. AFSCs have previously shown angiogenic potential and may present an alternative cell source for endothelial-like cells that could be used in range of applications, including the pre-vascularisation of TE constructs and the treatment of ischaemic diseases. This study investigated the ability of these cells to differentiate down an endothelial lineage with the aim of producing an endothelial-like cell suitable for use in pre-vascularisation. As hypoxia and the associated HIF-1 pathway have been implicated in the induction of angiogenesis in a number of biological processes, it was hypothesised that culture in hypoxic conditions could enhance the endothelial differentiation of AFSCs. The cells were cultured in endothelial cell media supplemented with 50 ng mL(-1) of VEGF, maintained in normoxia, intermittent hypoxia or continuous hypoxia and assessed for markers of endothelial differentiation at day 7 and 14. The results demonstrated that AFSCs subjected to these culture conditions display an endothelial gene expression profile and adopted functional endothelial cell characteristics indicative of early endothelial differentiation. Culture in continuous hypoxia enhanced endothelial gene expression but did not enhance functional endothelial cell characteristics. Overall, AFSCs subjected to endothelial stimuli demonstrated a less mature endothelial gene expression profile and phenotype when compared with HUVECs, the endothelial cell control. However, this study is the first time that the positive effect of an extended period of continuous hypoxic culture on endothelial differentiation in AFSCs has been demonstrated.