Targeting Multiple Hallmarks of Skin Aging: Preclinical and Clinical Efficacy of a Novel Growth Factor-Based Skin Care Serum.

INTRODUCTION: The aging process involves numerous biological mechanisms that have been characterized and proposed as the "hallmarks of aging." Targeting the processes and pathways related to these hallmarks of aging that cause and promote skin aging could provide anti-aging benefits. A novel topical growth factor-based skin care serum (A+) was developed using human fibroblast conditioned media. This study aimed to assess the effects of A+ on four hallmarks of aging and its clinical efficacy in skin rejuvenation in subjects with moderate to severe overall facial photodamage. METHODS: Preclinical studies included immunohistochemistry in human ex vivo skin, and gene expression analysis in human 3D skin models. A 24-week, vehicle placebo-controlled study, including FaceQ patient-reported outcomes and skin biopsy analysis, was performed to assess clinical efficacy and tolerability. RESULTS: Treatment with A+ resulted in reduced expression of cell senescence biomarker H2A.J and upregulation of genes associated with proteasome, autophagy, stemness, and intercellular communication. Clinical assessments showed A+ provided significantly greater reductions in sagging, coarse lines/wrinkles, fine lines/wrinkles, overall photodamage, and overall hyperpigmentation compared with placebo. Subjects felt they appeared younger-looking, reporting a median decrease in self-perceived age of 6 years after 12 weeks of use. Decreased levels of H2A.J and increased expression of key dermal extracellular matrix and epidermal barrier components, including collagen and elastin, were observed in skin biopsy samples. CONCLUSION: The present study shows for the first time the potential effects of a topical growth factor-based cosmeceutical on cellular processes related to four hallmarks of aging (cellular senescence, loss of proteostasis, stem cell exhaustion, and altered intercellular communication) to help delay the aging process and restore aged skin. A+ targets the biological mechanisms underlying the aging process itself and stimulates skin regeneration, resulting in rapid and significant clinical improvements.

Upregulation of Extracellular Matrix Genes Corroborates Clinical Efcacy of Human Fibroblast-Derived Growth Factors in Skin Rejuvenation.

Skin care products may use various active ingredients to support skin rejuvenation including growth factors and other molecules that help to regenerate extracellular matrix (ECM) and promote skin repair. The biological effect of skin care products with a strong anti-aging claim was assessed in gene expression analyses using an in vitro human skin model. Application of products containing human fibroblast-derived growth factors resulted in signifcant upregulation of genes encoding ECM components including collagens and elastin. Human fibroblasts cultured under hypoxic conditions show increased gene expression of stem cell markers, and their conditioned media could possibly further support skin rejuvenation. Furthermore, a double-blind, randomized, placebo-controlled study was con-ducted in subjects with moderate to severe facial photodamage to assess the cosmetic clinical efficacy of a product containing human fibroblast-derived growth factors. The test product group demonstrated significantly greater reductions in the appearance of fne lines/wrinkles, coarse line/wrinkles, and overall photodamage, compared to the placebo group. Altogether, the results suggest that human fibroblast-derived growth factors support skin rejuvenation by stimulating dermal fibroblasts to generate ECM.

Effects of Human Fibroblast-Derived Extracellular Matrix on Mesenchymal Stem Cells.

Stem cell fate is largely determined by the microenvironment called niche. The extracellular matrix (ECM), as a key component in the niche, is responsible for maintaining structural stability and regulating cell proliferation, differentiation, migration and other cellular activities. Each tissue has a unique ECM composition for its needs. Here we investigated the effect of a bioengineered human dermal fibroblast-derived ECM (hECM) on the regulation of human mesenchymal stem cell (hMSC) proliferation and multilineage differentiation. Human MSCs were maintained on hECM for two passages followed by the analysis of mRNA expression levels of potency- and lineage-specific markers to determine the capacity of MSC stemness and differentiation, respectively. Mesenchymal stem cells pre-cultured with or without hECM were then induced and analyzed for osteogenesis, adipogenesis and chondrogenesis. Our results showed that compared to MSCs maintained on control culture plates without hECM coating, cells on hECM-coated plates proliferated more rapidly with a higher percentage of cells in S phase of the cell cycle, resulting in an increase in the CD90+/CD105+/CD73+/CD45- subpopulation. In addition, hECM downregulated osteogenesis and adipogenesis of hMSCs but significantly upregulated chondrogenesis with increased production of collagen type 2. In sum, our findings suggest that hECM may be used to culture hMSCs for the application of cartilage tissue engineering.

Evolution of a Biosynthetic Temporary Skin Substitute: A Preliminary Study.

OBJECTIVE: To compare PermeaDerm to first temporary biosynthetic skin substitute (Biobrane, cleared by the Food and Drug Administration in 1979). METHODS: Different temporary skin substitutes (Biobrane, PermeaDerm, and PermeaDerm derivatives) were tested for physical differences, impact on healing wounds, inflammatory response, and ability to allow adequate growth of dermal fibroblasts and mesenchymal stem cells without accumulation of excessive scar-forming myofibroblasts. Proliferation of fibroblasts and stem cells on various skin substitutes was measured, and myofibroblast marker accumulation was evaluated by the expression of α-smooth muscle actin and fibronectin. Fibroblast migration was measured by tracking viable cells with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye. RESULTS: In vivo testing shows PermeaDerm works well as a temporary skin substitute, performing better than Biobrane with respect to inflammation and fluid accumulation. Tissue culture techniques revealed that cells on PermeaDerm grow in a more uniform fashion and migrated to a greater extent than cells on Biobrane. Furthermore, cells grown in the presence of PermeaDerm expressed lower levels of the myofibroblast markers α-smooth muscle actin and fibronectin than cells grown on Biobrane. CONCLUSION: PermeaDerm with variable porosity possesses all attributes and properties known to be important for a successful temporary skin substitute and enables the clinician to control porosity from essentially zero to what the wound requires. The ability of the clinician to minimize wound desiccation without fluid accumulation is related to the reduction of punctate scarring.

Inhibition of metastasis of circulating human prostate cancer cells in the chick embryo by an extracellular matrix produced by foreskin fibroblasts in culture.

We have previously demonstrated the increased metastatic potential of human prostate cancer circulating tumor cells (CTC), compared to their parental cells, in both orthotopic mouse models and the chick embryo model. In the current study, we asked whether an extracellular matrix (ECM), produced by human foreskin fibroblasts in culture, could inhibit PC-3 human prostate cancer CTC metastasis in the chick embryo model. The chorioallantoic membranes (CAM) of 18 chicken embryos were inoculated with either PC-3 human prostate cancer cells or PC-3 CTCs, both stably expressing green fluorescent protein (GFP). Embryos were divided into six groups: PC-3 parental-cell control; PC-3 plus soluble ECM; PC-3 parental cells plus semi-solid ECM; PC-3 CTC control; PC-3 CTC plus soluble ECM, and PC-3 CTC plus semi-solid ECM. Twelve hours following inoculation of the cells, a single dose of 100 µl of either soluble or semi-solid ECM was added to the appropriate group. Embryo brains were removed on day 8 post-inoculation, and were processed for cryosectioning. Imaging was performed on the cryosections using a scanning laser microscope in order to count metastatic foci. PC-3 controls had an average of 11.1 metastatic foci compared to 2.55 in the PC-3 plus soluble ECM group and 2.76 (p<0.0001) in the PC-3 plus semi-solid ECM group (p<0.0001). ECM treatment had even greater efficacy on the CTC cells, with an average of 30.9 metastatic foci in the CTC controls compared to 4.38 in the CTC plus soluble ECM group (p<0.0001) and 4.18 in the CTC plus semi-solid ECM group (p<0.0001). The results demonstrate that reduction of CTC metastatic potential is possible, in this case with an ECM produced by human foreskin fibroblasts in culture.

Hair regrowth following a Wnt- and follistatin containing treatment: safety and efficacy in a first-in-man phase 1 clinical trial.

Research has shown the importance of follistatin, Wnt 7a, and wound healing growth factors on the stimulation of bulge cells and inter-follicular stem cells to induce hair growth. We have studied the effects of a bioengineered, non-recombinant, human cell-derived formulation, termed Hair Stimulating Complex (HSC), containing these factors to assess its hair growth activity in male pattern baldness. HSC showed in vitro Wnt activity and contained follistatin, KGF, and VEGF. The clinical study was a double-blind, placebo-controlled, randomized single site trial and was designed to evaluate safety of the HSC product and assess efficacy in stimulating hair growth. All 26 subjects tolerated the single, intradermal injection of HSC procedures well, and no signs of an adverse reaction were reported. Histopathological evaluation of the treatment site biopsies taken at 22 and 52 weeks post-treatment revealed no abnormal morphology, hamartomas, or other pathological responses. Trichoscan image analysis of HSC-treated sites at 12 and 52 weeks showed significant improvements in hair growth over the placebo. At the initial 12-week evaluation period, HSC-treated sites demonstrated an increase in hair shaft thickness (6.3%±2.5% vs. -0.63%±2.1%; P=0.046), thickness density (12.8%±4.5% vs. -0.2%±2.9%; P=0.028), and terminal hair density (20.6±4.9% vs. 4.4±4.9%; P=0.029). At one year, a statistically significant increase in total hair count (P=0.032) continued to be seen. These results demonstrate that a single intradermal administration of HSC improved hair growth in subjects with androgenetic alopecia and is a clinical substantiation of previous preclinical research with Wnts, follistatin, and other growth factors associated with wound healing and regeneration.

Three-dimensional fibroblast cultures stimulate improved ventricular performance in chronically ischemic canine hearts.

The current study's purpose was to evaluate the safety and biological effect of a scaffold-based three-dimensional human dermal fibroblast culture (3DFC, also known as Anginera™) to treat chronically ischemic canine hearts. It was hypothesized that treatment with 3DFC would be safe and significantly improve ventricular performance and wall motion. In this study, chronic myocardial ischemia was induced in 40 animals through the surgical placement of an ameroid constrictor. Approximately 30 days after ameroid placement, animals were randomized into four test groups: (1) sham treatment, (2) one unit of acellular 3DFC, (3) one unit of viable 3DFC, and (4) three units of viable 3DFC. Animals were necropsied 30 or 90 days after treatment. Evaluation of the safety endpoint demonstrated the safety of 3DFC at all dosing levels and at both time points. Additionally, parameters of cardiac output, left ventricular ejection fraction, left ventricular end systolic volume index, and systolic wall thickening support the conclusions that 3DFC stimulates a positive biologic effect on ischemic canine hearts. Further, these data support the conclusion that treatment with viable 3DFC improves ventricular performance and ventricular wall motion in chronically ischemic canine hearts 30 days after treatment.

Human Embryonic-like ECM (hECM) Stimulates Proliferation and Differentiation in Stem Cells While Killing Cancer Cells.

BACKGROUND AND OBJECTIVES: There are a number of unique processes seen in the developing fetus that cease post-partum including that tumors rarely form, and scar-less wound healing and digit regeneration occur. In addition, cancer lines have been "reprogrammed" by co-culture with embryonic extracellular matrix (ECM). METHODS AND RESULTS: We have developed a naturally secreted human ECM (hECM) with embryonic-like characteristics which is secreted by neonatal fibroblasts grown in microcarrier suspension cultures under hypoxia. This upregulates a number of substances associated with stem cell niches in the body including various laminins, Collagen 4, CXCL12, NID1, NID2, and NOTCH2. hECM has been shown to support proliferation of hESCs and MSCs and diminish or eliminate tumor load in melanoma (B16), adenocarcinoma (MDA-MB-435), colon cancer (HT29) and glioma (C6) in both in vitro and in vivo animal studies. In the tumor chorioallantoic membrane (tumcam) model hECM significantly inhibited tumor growth and in subcutaneous mouse xenograft experiments, tumor growth was inhibited from 70∼90%. Co-cultures of fibroblasts and mesothelioma show support of fibroblast expansion with a concurrent inhibition of mesothelioma. The inhibitory affect is selective for cancer cells and cancer stem cells through the upregulation of Caspase 9 which forces the cells into apoptosis. CONCLUSIONS: These data show that hECM has the potential to show benefit in the treatment of various cancers as a coating for biopsy needle, tissue filler post tumor removal, and as an injectable into the tumor site.

Hypoxic conditioned culture medium from fibroblasts grown under embryonic-like conditions supports healing following post-laser resurfacing.

OBJECTIVES: Treatment of facial skin perturbed by laser resurfacing with a novel, topical hypoxic conditioned culture medium (HCCM) product results in apparent, accelerated wound recovery time. The HCCM product is conditioned by neonatal fibroblasts under hypoxic conditions and used as the active ingredient in a formulated topical lotion. The HCCM contains significant quantities of growth factors such as vascular endothelial growth factor, keratinocyte growth factor, and interleukin-8. As these molecules are known to play an important role in normal wound healing in vivo, we conducted a pilot clinical evaluation "Proof of Concept" in which individuals, after receiving laser resurfacing, were instructed to use either active or placebo lotion on their abraded skin. METHODS: The end points used were clinical assessment of the time to complete healing, clinical and bioinstrumental mexameter measurements of erythema, and the number of days of rescue petrolatum use by patients, post-laser. RESULTS: Day 7, post-laser treatment, resulted in a greater improvement in erythema, and re-epithelization of the peri-oral and peri-ocular regions in subjects using the active lotion vs. placebo control as determined by blinded, clinical evaluation of gross photographs and bioinstrumental mexameter measurements. A statistically significant reduction in rescue petrolatum use in active lotion-treated subjects was reported. Finally, no attendant cutaneous safety concerns (e.g., irritant/allergic dermatitis) were reported with either active or placebo lotion. CONCLUSIONS: This HCCM product may have broad applications within the field of skin wound repair.

Cardiac patch constructed from human fibroblasts attenuates reduction in cardiac function after acute infarct.

The current experiments used a scaffold-based, three-dimensional, human dermal fibroblast culture (3DFC) as a cardiac patch to stimulate revascularization and preserve left ventricular (LV) function of the infarcted LV in severe combined immunodeficient (SCID) mice. The 3DFC contains viable cells that secrete angiogenic growth factors and has been previously shown to stimulate angiogenesis. The hypothesis tested was that a 3DFC cardiac patch would attenuate a reduction in LV function of infarcted hearts. Five groups of mice were studied, including normal SCID mice (n = 13), normal SCID mice with 3DFC (n = 6), infarcted SCID mice (n = 6), infarcted mice with nonviable 3DFC (n = 6), and infarcted SCID mice with 3DFC (n = 6). An occlusion of a branch of the left anterior descending (LAD) coronary artery was performed by thermal ligation, and 3DFC was sized to the damaged area and implanted onto the epicardium at the site of tissue injury. Fourteen days postsurgery, LV mechanics were characterized with the Millar conductance catheter system (CCS). The data demonstrated that 3DFC-treated infarcted myocardium had significantly higher ejection fractions (EFs) compared with infarct-only mice (58.9 +/- 10.8 versus 31.0 +/- 5.8%, respectively; p < 0.05). Preload recruitable stroke work (PRSW) parameters were significantly higher in 3DFC-treated mice compared with infarct-only mice (64.6 +/- 11.9 versus 36.8 +/- 6.4 mmHg, respectively; p < 0.05). These results show that the 3DFC as a cardiac patch functioned to attenuate further loss of LV function accompanying acute myocardial infarct and that this may be related in part to myocardial revascularization.

From lab bench to market: critical issues in tissue engineering.

Revolutionary advances in tissue engineering are redefining approaches to tissue repair and transplantation through the creation of replacement tissues that remain biointeractive after implantation, imparting physiologic functions as well as structure to the tissue or organ damaged by disease or trauma.(1,2) Over the last decade this field has moved from "science fiction" to "science fact" with the research-oriented acceptance of its potential to regulatory approvals allowing commercial products to be available for use in many countries. The maintenance of tissue integrity, functionality, and viability from cell seeding through product manufacture, shipping, and end-use has been accomplished through innovations in design and scale-up of both tissue growth and preservation processes. These unique systems have enabled the delivery of tissue-engineered products that are uniform inter- and intra-lot, readily available as off-the-shelf products, easy to use, and efficacious. Skin replacement products are the most advanced, with several tissue-engineered wound care materials on the market in the U.S. and in several international communities.(3-5) The potential impact of this field is far broader, offering novel solutions to the medical field for drug screening and development, genetic engineering, and total tissue and organ replacement.