Bone Marrow Mesenchymal Stem Cells to Heal Chronic Diabetic Wounds
Diabetic foot ulcers (DFU), chronic, non-healing wounds on the feet of diabetic patients, present a serious challenge to global health. These ulcers affect between 15-25% of the 18-21 million Americans who have diabetes (world-wide incidence of diabetes: 366 million people). DFUs have a huge impact on our health care system, not only in terms of economic cost, but also from a psychosocial perspective, associated with significant morbidities, decrease in quality of life, prolonged hospitalization and importantly, often result in the amputation loss of lower extremity. In the United States, persons with diabetes are at twice the risk for amputation compared to non-diabetic individuals. According to recent census, DFU is the leading cause of lower limb amputation and greater than 85% of amputations are preceded by an active foot ulcer.
Treatments for curing DFU are very far from optimal. Current standard of care can cure only about 30% of DFU and even the most advanced therapies, cell-based devices containing skin-derived keratinocytes and fibroblasts, boost the cure rate only to about 50%, leaving a tremendous unmet need for new effective cures for DFU.
The research that we propose with our collaborative partners in Germany is directed specifically at this problem. The candidate device is a combination of mesenchymal stem cells that have curative powers, and secrete potent stimulatory molecules, coupled with a collagen scaffolding creating a template upon which new tissue can be rebuilt and regenerated. The combined mesenchymal stem cell- scaffold device will be pre-conditioned so that its reparative properties are maximized. Testing of the material will occur in animal models that closely mimic the human DFU condition, so that the results can be reliably translated to a human curative product. The product will come to the clinic as living mesenchymal stem cells embedded in the pre-optimized scaffolding. All the treating physician will need to do is rinse the bandage-like material and apply it to the wound. Based on our preliminary studies that have examined the potent healing and revascularizing effects of MSC on damaged tissues, we anticipate that rapid healing will ensue.
While the number of individuals with all forms of chronic wounds is increasing in the general population, particularly with the rise of diabetes and aging of the population, the number of individuals affected by diabetic foot ulcers (DFU), the target disease for the development candidate in this proposal, is increasing in California at an alarming rate. That is because the prevalence of type 2 diabetes is now increasing within the state of California to epidemic proportions. In 2002, over one million California adults age 45 and older were diagnosed with diabetes, and by 2005 that number had risen to 1.5 million: 5.9% of the California population. For reasons that are not all that clear, there are marked differences in the prevalence of diabetes in different Californian ethnic and racial groups. Among Californians 65 and older, diabetes is significantly more common in African Americans (25.6%) , and Latinos ( 24.4%) as compared to caucasians (12.2%). (1) The diabetes brings with it devastating health impacts: it is the sixth most common cause of death in the United States. Among the morbidities associated with diabetes, DFU is one of the most debilitating. Approximately 15-25 percent of patients with diabetes will develop DFU, and of those, six percent will be hospitalized due to infection or other ulcer-related complication. According to a recent census, DFU is the leading cause of lower limb amputation and greater than 85% of amputations are preceded by an active foot ulcer.
Sadly for our state, we lead others in the US in the prevalence of DFU: "Of the 45 areas (44 states and DC) that reported information from the BRFSS diabetes module, Indiana (16.3%), California (16.2%), and Nevada (16.2%) had the highest age-adjusted prevalence of a history of foot ulcer among persons with diabetes, and Colorado (7.4%), Wisconsin (8.8%), and Hawaii (8.9%) had the lowest " (2).
Treatments for curing DFU are very far from optimal. Current standard of care can cure only about 30% of DFU and even the most advanced therapies, cell-based devices containing skin derived keratinocytes and fibroblasts, boosts the cure rate only to about 50%, leaving a tremendous unmet need for new effective cures for DFU, particularly in California. We anticipate that the development candidate that we propose, a stem cell-based “biological bandage”, will bring such a new and effective cure to our citizens who are suffering from diabetic foot ulcers.
Sources: 1) California Health Care Survey, UCLA, http://www.chis.ucla.edu/
2) CDC reports Morbidity and Mortality Weekly Report (MMWR), http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5245a3.htm
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.
This early translational award is focused on creating a stem cell based therapy to improve healing in diabetic foot ulcers (DFU). DFU are probably the major complication of diabetes mellitus. It is estimated that between 15- 25 % of all patients who have diabetes will develop this complication, and this goes on to be the cause of an astounding 85% of all lower leg amputations. The incidence of diabetes is increasing worldwide, and sadly, California leads the US with the highest prevalence of DFU. New therapies using live cells have been marketed as major advances in the therapy of this devastating problem, but their success is limited. Here we have proposed to engineer a wound replacement tissue that is laden with bone marrow derived mesenchymal stem cells (MSC), that have been pre-treated using conditions that will optimize their ability to repair the wound.
In this funding period (year 2) we have met all the preset milestones for this work. In particular, we have developed a model of impaired skin wound healing in a diabetic mouse, and demonstrated that our combination therapy of preconditioning the MSC with a treatment of hypoxia (low oxygen tension) and a drug commonly used to block the beta adrenergic receptor can improve healing in this impaired healing model. The MSC are pretreated and then adhered to an extracellular matrix that forms a scaffold for cell attachment and tissue regeneration. Improvement of over 20% of healing rates have been achieved in this model. A second milestone of this year’s work was to initiate a model of impaired healing in pig skin, as this mirrors human skin wound healing much more closely. We show the development of this model, how multiple healing parameters are measured in this model, and how these metrics can show us the impairment of healing that occurs when the wound is infected with common wound pathogenic bacteria. Our plan is to test our pre-conditioned MSC/matrix device in these two models and to not only demonstrate efficacy, but also to understand the mechanisms by which the device improves healing. At the end of this three year funding period we hope to have sufficient data to lead to an IND submission to the FDA for initiation of a clinical trial in patients with DFU.
Bone Marrow Mesenchymal Stem Cells to Heal Chronic Diabetic Wounds
Our goal was to engineer a device to improve healing in diabetic foot ulcers, a devastating consequence of diabetes that occurs in about 25% of all diabetic patients and is responsible for most leg or foot amputations. More than 3 million people in the US and up to 91 million people worldwide have diabetic foot ulcers (DFU). There is a clear medical need. There are products on the market that can improve wound healing for some, but not all patients. This causes a large financial burden for the health care system, and great suffering for the patients who live with open wounds, often infected, that progress to amputations. Therefore there is a clear medical need for advanced therapies to heal diabetic ulcers faster.
We proposed to create a device consisting of a scaffold for dermal regeneration (SDR) populated with mesenchymal stem cells (MSC) that have been pre-conditioned for optimized reparative function. We formed a team of established wound and stem cell/matrix experts, and this team has indeed successfully engineered and demonstrated efficacy of the device in two animal models, and is now ready to progress to IND-enabling studies in support of our very promising Development Candidate.
During this Early Translational grant, we developed a product that consists of an FDA-approved scaffold for dermal regeneration (SDR) filled with human bone marrow-derived Mesenchymal Stem Cells (MSC). These are then pre-incubated for 2 days in hypoxia and in the presence of a beta adrenergic antagonist. We have completed studies that demonstrate that this “next generation” stem cell product is highly efficacious in healing skin wounds, using diabetic mouse models.
In this third year of funding, our top priority was to evaluate our product in a pig model for skin wound healing. Confirming our previous results in diabetic mice, we found that two weeks after administrating our product to the wounds of a Yucatan minipig, we achieved over a 20% higher rate of wound healing (re-epithelialization) as compared to SDR alone. These results are very promising, even more so because the improvement we note is in normal, healthy pigs, who ordinarily heal well. We anticipate that when we use this device on wounds that are infected, or in diabetic animals, where healing is delayed or impaired, the increase in healing rates will be even greater.
We are very excited about these results and hope to continue working on this project through future CIRM funding, in order to bring this highly promising therapeutic to a clinical trial.
- J Cell Biochem (2015) Mesenchymal Stem Cells Respond to Hypoxia by Increasing Diacylglycerols. (PubMed: 26212931)
- Stem Cells (2015) Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo. (PubMed: 25702874)
- Wound Repair Regen (2015) Importance of defining experimental conditions in a mouse excisional wound model. (PubMed: 25703258)
- Wound Repair Regen (2014) Full-thickness splinted skin wound healing models in db/db and heterozygous mice: implications for wound healing impairment. (PubMed: 24844336)
- Adv Wound Care (New Rochelle) (2014) Epithelialization in Wound Healing: A Comprehensive Review. (PubMed: 25032064)
- Am J Clin Dermatol (2014) Diabetic foot ulcer: an evidence-based treatment update. (PubMed: 24902659)
- PLoS One (2014) PDGF-BB does not accelerate healing in diabetic mice with splinted skin wounds. (PubMed: 25121729)
- Stem Cells Transl Med (2014) Crosstalk Between Adrenergic and Toll-Like Receptors in Human Mesenchymal Stem Cells and Keratinocytes: A Recipe for Impaired Wound Healing. (PubMed: 24760207)
- Stem Cells (2011) Effects on Proliferation and Differentiation of Multipotent Bone Marrow Stromal Cells Engineered to Express Growth Factors for Combined Cell and Gene Therapy. (PubMed: 21898687)
- Stem Cells (2011) Induced Pluripotent Stem Cell - Derived Mesenchymal Stem Cells: Progress Toward Safe Clinical Products. (PubMed: 21898694)