Robert C. Rennert

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme‐linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet‐derived growth factor‐AA (PDGF‐AA), PDGF‐BB, transforming growth factor α (TGFα), TGFβ1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony‐stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL‐4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications.

Stem cell recruitment after injury: lessons for regenerative medicine.

Tissue repair and regeneration are thought to involve resident cell proliferation as well as the selective recruitment of circulating stem and progenitor cell populations through complex signaling cascades. Many of these recruited cells originate from the bone marrow, and specific subpopulations of bone marrow cells have been isolated and used to augment adult tissue regeneration in preclinical models. Clinical studies of cell-based therapies have reported mixed results, however, and a variety of approaches to enhance the regenerative capacity of stem cell therapies are being developed based on emerging insights into the mechanisms of progenitor cell biology and recruitment following injury. This article discusses the function and mechanisms of recruitment of important bone marrow-derived stem and progenitor cell populations following injury, as well as the emerging therapeutic applications targeting these cells.

Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells

Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations

IntroductionPathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.MethodsIn this study, we examined the impact of diabetes on the murine ASC niche as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single-cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.ResultsOur results demonstrate that diabetes alters the ASC niche in situ and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single-cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.ConclusionsPerturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in patients with diabetes may be limited and that interventions to improve cell function before application are warranted.

Mechanotransduction and fibrosis

Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

Tissue Engineering and Regenerative Repair in Wound Healing

Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

Tracking the Elusive Fibrocyte: Identification and Characterization of Collagen-Producing Hematopoietic Lineage Cells During Murine Wound Healing

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single‐cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45− and Cd11b−, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing. Stem Cells 2014;32:1347–1360

Isolation of Human Adipose-Derived Stromal Cells Using Laser-Assisted Liposuction and Their Therapeutic Potential in Regenerative Medicine

Harvesting adipose‐derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third‐generation ultrasound‐assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser‐assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser‐assisted lipoaspirate and suction‐assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical‐sized cranial defect in athymic nude mice. Although ASCs isolated from suction‐assisted lipoaspirate and laser‐assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34+CD31−CD45−) in the stromal vascular fraction were all significantly less with laser‐assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro‐computed tomography revealed significantly more healing with ASCs isolated from suction‐assisted lipoaspirate relative to laser‐assisted lipoaspirate at the 4‐, 6‐, and 8‐week time points (p < .05). Therefore, as laser‐assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.

Transdermal deferoxamine prevents pressure-induced diabetic ulcers

Significance Diabetes is the leading cause of nontraumatic amputations. There are no effective therapies to prevent diabetic ulcer formation and only modestly effective technologies to help with their healing. To enhance diabetic wound healing we designed a transdermal delivery system containing the FDA-approved small molecule deferoxamine, an iron chelator that increases defective hypoxia inducible factor-1 alpha transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. This system overcomes the challenge of delivering hydrophilic molecules through the normally impermeable stratum corneum and both prevents diabetic ulcer formation and improves the healing of existing diabetic wounds. This represents a prophylactic pharmacological agent to prevent ulcer formation that is rapidly translatable into the clinic and has the potential to ultimately transform the care and prevention of diabetic complications. There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Studies in fat grafting: Part IV. Adipose-derived stromal cell gene expression in cell-assisted lipotransfer.

Background: Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting. Methods: Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells. Results: Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein–positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting. Conclusions: Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.