Year 1

Overall, the progress can be summarized in the following aspects: (1) We will continue our project using bone marrow-derived MSC. (2) MSC associate, distribute and are viable within the scaffold for dermal regeneration (Integra™ Matrix, SDR), “bio-activating” the scaffold. (3) A murine diabetic impaired wound-healing model has been established. (4) Preconditioning by modulation of β–AR signals and hypoxia show a positive effect on MSC and MSC in SDR.

Our first goal was to determine whether to continue our project with bone marrow- (BM-) or adipose tissue- (AT-) derived mesenchymal stem cells (MSC). Here we show that both cell types show similar surface markers: CD45-, CD73+, CD90+, CD105+, but expression of the pericyte marker CD146 is higher in BM- than AT-MSC. When tested for their potential to differentiate toward adipogenic and osteogenic lineages, both BM-MSC and AT-MSC showed comparable capacity, as evidenced by Oil Red O and Alizarin Red S, which stain triglycerides and calcium deposition respectively. In terms of their angiogenic activity, we presented in our 6 month-progress report that both BM- and AT- MSC showed very similar potential to induce migration of endothelial cells (HUVEC) in vitro. Now we have also considered the following aspects: (1) Isolation and expansion protocols for BM-MSC are well standardized. Isolation of AT-MSC typically requires collagenase treatment, which may present adverse effects after implantation. Most importantly, BM-MSCs have been approved for administration as drugs by FDA-equivalent institutions in Canada and New Zealand. Since our goal is to make an off-the-shelf product that can be administered in the clinic, we need a banked allogeneic cell source and have all our standard operating procedures (SOPs) well established in our UC Davis Good Manufacturing Practice (GMP) facility for normal healthy donor-derived BM-MSC.

To determine the cell load-capacity of the scaffold for dermal regeneration (Integra Matrix®), BM-MSCs were seeded at various concentrations. The highest concentration tested was equivalent to almost 4 million cells per square centimeter of SDR and this was still not the maximal cell load capacity. Using confocal microscopy we observed that the cell distribution appears relatively homogenous within the SDR, but 85% of cells are concentrated in the upper half of the scaffold. It is therefore perhaps feasible to increase cell load capacity by addition of cells from both sides of the scaffold. However, at this point it is unclear what will be the optimal cell dose required, since this has to be established through functional studies in vivo with varying cell doses per SDR. These studies will continue through year 2 of funding.

We also quantified cell viability over time using different methods. We concluded that the cell number remains rather stable over at least 14 days, probably due to similar rates in cell death and proliferation. To evaluate the angiogenic potential of MSC in SDR in vitro, we performed HUVEC migration assays, with supernatants of either empty SDR or MSC-containing SDR in either 21% (normoxia) or 1% O2 (hypoxia). We observed that supernatant of both, MSC-containing SDR in control conditions and hypoxia induced migration of HUVEC. It also appears that hypoxia enhances the angiogenic potential of BM-MSC. In terms of modulation of β-AR signals in BM-MSC, now we report how both Epinephrine and the β-AR antagonist Timolol increase the osteogenic potential of BM-MSC but did not affect cell viability. From these experiments, we conclude that modulation of β-AR signals do not greatly affect MSC in SDR in vitro. However, significantly absent in these assays are a component of the wound – bacterial antigens that could activate TLR receptors on the surface of the MSC and thus alter the response to adrenergic signals. In the next year of funding we will examine this effect and their effect on tissue healing in vivo, since the effects could be observed in the damaged tissue.

Finally, we have established an impaired disease model in mice. To mimic the background of human patients with chronic wounds, we used diabetic animals. These mice present high glycemia levels. Most important, these animals present slower wound closure dynamics, strongly resembling the human condition. We have performed preliminary experiments testing whether human BM-MSC containing SDR will provide wound closure improvement. However, when compared to no treatment, we did not observe improvement, which may be related to a stenting function of the SDR placed in the wound. Ongoing experiments are comparing SDR to BM-MSC containing SDR, as well as alternative more flexible SDR.