Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.

Environmental manipulation to promote stem cell survival in vivo: use of aggregation, oxygen carrier, and BMP-2 co-delivery strategies.

While mesenchymal stem cell (MSC)-based strategies for critically-sized bone defect repair hold promise, poor cell survival in vivo remains a significant barrier to the translation of these therapeutics. One method employed to extend the survival of MSCs is the formation of three-dimensional aggregates, a strategy which modulates the immunomodulatory secretome of the cells, thereby influencing the local inflammatory environment and potentially bone tissue repair. Enrichment of cell-seeded hydrogels with oxygen carriers to counter the hypoxic conditions encountered in vivo or co-delivery of cells with growth factors, are also strategies employed to modulate the cell micro-environment. In this study, we examined the effect of human MSC (hMSC) and rat MSC (rMSC) aggregation on cell survival and bone tissue regeneration within both immunocompromised (nude) and syngeneic (Lewis) rat models. Despite a heightened release of paracrine factors from stem cell aggregates in vitro, the delivery of hMSC or rMSC aggregates in their respective rat models had no beneficial impact on cell survival, construct vascularization, or critically-sized bone defect repair. Co-delivery of oxygen carrier perfluorotributylamine (PFTBA) within the alginate hydrogel delivery vehicle impeded in vivo bone regeneration in both MSC-seeded and acellular constructs. Although rMSC seeding was observed to enhance the osteoinductive potential of bone morphogenetic protein 2 (BMP-2)-containing constructs in vitro, co-delivery of rMSC and BMP-2 to the femoral defect space attenuated bone repair in vivo compared to BMP-2 delivery alone. Overall, despite in vitro evidence to the contrary, the present study observed no beneficial effects of these delivery strategies on cell-based bone tissue repair.

Effects of in vitro endochondral priming and pre-vascularisation of human MSC cellular aggregates in vivo.

INTRODUCTION: During endochondral ossification, both the production of a cartilage template and the subsequent vascularisation of that template are essential precursors to bone tissue formation. Recent studies have found the application of both chondrogenic and vascular priming of mesenchymal stem cells (MSCs) enhanced the mineralisation potential of MSCs in vitro whilst also allowing for immature vessel formation. However, the in vivo viability, vascularisation and mineralisation potential of MSC aggregates that have been pre-conditioned in vitro by a combination of chondrogenic and vascular priming, has yet to be established. In this study, we test the hypothesis that a tissue regeneration approach that incorporates both chondrogenic priming of MSCs, to first form a cartilage template, and subsequent pre-vascularisation of the cartilage constructs, by co-culture with human umbilical vein endothelial cells (HUVECs) in vitro, will improve vessel infiltration and thus mineral formation once implanted in vivo. METHODS: Human MSCs were chondrogenically primed for 21 days, after which they were co-cultured with MSCs and HUVECs and cultured in endothelial growth medium for another 21 days. These aggregates were then implanted subcutaneously in nude rats for 4 weeks. We used a combination of bioluminescent imaging, microcomputed tomography, histology (Masson's trichrome and Alizarin Red) and immunohistochemistry (CD31, CD146, and α-smooth actin) to assess the vascularisation and mineralisation potential of these MSC aggregates in vivo. RESULTS: Pre-vascularised cartilaginous aggregates were found to have mature endogenous vessels (indicated by α-smooth muscle actin walls and erythrocytes) after 4 weeks subcutaneous implantation, and also viable human MSCs (detected by bioluminescent imaging) 21 days after subcutaneous implantation. In contrast, aggregates that were not pre-vascularised had no vessels within the aggregate interior and human MSCs did not remain viable beyond 14 days. Interestingly, the pre-vascularised cartilaginous aggregates were also the only group to have mineralised nodules within the cellular aggregates, whereas mineralisation occurred in the alginate surrounding the aggregates for all other groups. CONCLUSIONS: Taken together these results indicate that a combined chondrogenic priming and pre-vascularisation approach for in vitro culture of MSC aggregates shows enhanced vessel formation and increased mineralisation within the cellular aggregate when implanted subcutaneously in vivo.

Functional augmentation of naturally-derived materials for tissue regeneration.

Tissue engineering strategies have utilized a wide spectrum of synthetic and naturally-derived scaffold materials. Synthetic scaffolds are better defined and offer the ability to precisely and reproducibly control their properties, while naturally-derived scaffolds typically have inherent biological and structural properties that may facilitate tissue growth and remodeling. More recently, efforts to design optimized biomaterial scaffolds have blurred the line between these two approaches. Naturally-derived scaffolds can be engineered through the manipulation of intrinsic properties of the pre-existing backbone (e.g., structural properties), as well as the addition of controllable functional components (e.g., biological properties). Chemical and physical processing techniques used to modify structural properties of synthetic scaffolds have been tailored and applied to naturally-derived materials. Such strategies include manipulation of mechanical properties, degradation, and porosity. Furthermore, biofunctional augmentation of natural scaffolds via incorporation of exogenous cells, proteins, peptides, or genes has been shown to enhance functional regeneration over endogenous response to the material itself. Moving forward, the regenerative mode of action of naturally-derived materials requires additional investigation. Elucidating such mechanisms will allow for the determination of critical design parameters to further enhance efficacy and capitalize on the full potential of naturally-derived scaffolds.

In vivo bioluminescent tracking of mesenchymal stem cells within large hydrogel constructs.

The use of multicomponent scaffolds for cell implantation has necessitated sophisticated techniques for tracking of cell survival in vivo. Bioluminescent imaging (BLI) has emerged as a noninvasive tool for evaluating the therapeutic potential of cell-based tissue engineering strategies. However, the ability to use BLI measurements to longitudinally assess large 3D cellular constructs in vivo and the effects of potential confounding factors are poorly understood. In this study, luciferase-expressing human mesenchymal stem cells (hMSCs) were delivered subcutaneously within agarose and RGD-functionalized alginate hydrogel vehicles to investigate the impact of construct composition and tissue formation on BLI signal. Results showed that alginate constructs exhibited twofold greater BLI counts than agarose constructs at comparable hMSC doses. However, each hydrogel type produced a linear correlation between BLI counts and live cell number, indicating that within a given material, relative differences in cell number could be accurately assessed at early time points. The survival efficiency of delivered hMSCs was highest for the lower cell doses embedded within alginate matrix. BLI signal remained predictive of live cell number through 1 week in vivo, although the strength of correlation decreased over time. Irrespective of hydrogel type or initial hMSC seeding dose, all constructs demonstrated a degree of vascularization and development of a fibrotic capsule after 1 week. Formation of tissue within and adjacent to the constructs was accompanied by an attenuation of BLI signal during the initial period of the image acquisition time-frame. In alginate constructs only, greater vessel volume led to a delayed rise in BLI signal following luciferin delivery. This study identified vascular and fibrotic tissue ingrowth as potential confounding variables for longitudinal BLI studies. Further investigation into the complexities of noninvasive BLI data acquisition from multicomponent constructs, following implantation and subsequent tissue formation, is warranted.