Bioengineered Self-assembled Skin as an Alternative to Skin Grafts.

For patients with extensive burns or donor site scarring, the limited availability of autologous and the inevitable rejection of allogeneic skin drive the need for new alternatives. Existing engineered biologic and synthetic skin analogs serve as temporary coverage until sufficient autologous skin is available. Here we report successful engraftment of a self-assembled bilayered skin construct derived from autologous skin punch biopsies in a porcine model. Dermal fibroblasts were stimulated to produce an extracellular matrix and were then seeded with epidermal progenitor cells to generate an epidermis. Autologous constructs were grafted onto partial- and full-thickness wounds. By gross examination and histology, skin construct vascularization and healing were comparable to autologous skin grafts and were superior to an autologous bilayered living cellular construct fabricated with fibroblasts cast in bovine collagen. This is the first demonstration of spontaneous vascularization and permanent engraftment of a self-assembled bilayered bioengineered skin that could supplement existing methods of reconstruction.

The importance of both fibroblasts and keratinocytes in a bilayered living cellular construct used in wound healing.

Cross talk between fibroblasts and keratinocytes, which maintains skin homeostasis, is disrupted in chronic wounds. For venous leg ulcers and diabetic foot ulcers, a bilayered living cellular construct (BLCC), containing both fibroblasts and keratinocytes that participate in cross talk, is a safe and effective product in healing chronic wounds. To show the importance of both cell types in BLCC, constructs were generated containing only fibroblasts or only keratinocytes and compared directly to BLCC via histology, mechanical testing, gene/protein analysis, and angiogenesis assays. BLCC contained a fully differentiated epithelium and showed greater tensile strength compared with one-cell-type constructs, most likely due to formation of intact basement membrane and well-established stratum corneum in BLCC. Furthermore, expression of important wound healing genes, cytokines, and growth factors was modulated by the cells in BLCC compared with constructs containing only one cell type. Finally, conditioned medium from BLCC promoted greater endothelial network formation compared with media from one-cell-type constructs. Overall, this study characterized a commercially available wound healing product and showed that the presence of both fibroblasts and keratinocytes in BLCC contributed to epithelial stratification, greater tensile strength, modulation of cytokine and growth factor expression, and increased angiogenic properties compared with constructs containing fibroblasts or keratinocytes alone.

Hair follicle neogenesis induced by cultured human scalp dermal papilla cells.

AIM: To develop a method by which human hair follicle dermal papilla (DP) cells can be expanded in vitro while preserving their hair-inductive potential for use in follicular cell implantation, a cellular therapy for the treatment of hair loss. MATERIALS & METHODS: DP cells were isolated from scalp hair follicles in biopsies from human donors. DP cell cultures were established under conditions that preserved their hair-inductive potential and allowed for significant expansion. The hair-inductive potential of cells cultured for approximately 36 doublings was tested in an in vivo flap-graft model. In some experiments, DiI was used to label cells prior to grafting. RESULTS: Under the culture conditions developed, cultures established from numerous donors reproducibly resulted in an expansion that averaged approximately five population doublings per passage. Furthermore, the cells consistently induced hair formation in an in vivo graft assay. Grafted DP cells appeared in DP structures of newly formed hairs, as well as in the dermal sheath and in the dermis surrounding follicles. Induced hair follicles persisted and regrew after being plucked 11 months after grafting. CONCLUSION: A process for the propagation of human DP cells has been developed that provides significant expansion of cells and maintenance of their hair-inductive capability, overcoming a major technical obstacle in the development of follicular cell implantation as a treatment for hair loss.

Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-gamma-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.

Safety and feasibility of percutaneous autologous skeletal myoblast transplantation in the coil-infarcted swine myocardium.

INTRODUCTION: Autologous skeletal myoblast transplantation (ASMT) for myocardial regeneration is a promising new treatment for patients with congestive heart failure secondary to myocardial infarction (MI). However, non-surgical delivery could broaden the utility of this approach. The present study was designed to evaluate the safety and feasibility of transplanting autologous skeletal myoblast (ASM) via endovascular delivery into the infarcted swine myocardium. METHODS: Seven female Yorkshire swine successfully underwent induced left ventricular MI. ASM biopsies were obtained from the hind limb of each animal and myoblasts were expanded in vitro. In a pilot experiment, ASM were labeled with iridium and short-term retention and biodistribution was determined 2 h after ASM delivery via the MyoStar needle-injection catheter inserted through the femoral artery. At 30 days post-infarction, the remaining animals were divided into three groups containing 2 animals each for percutaneous catheter delivery into the infarcted zone: group 1 control animals were injected with media only, group 2 and 3 animals were injected with approximately 300 x 10(6) and 600 x 10(6) ASM, respectively. Sixty days post-transplantation, the swine hearts were harvested. RESULTS: During the 60-day period between transplantation and harvest, no adverse events were recorded, and continuous rhythm monitoring revealed no arrhythmias. In the small sampling size, myocardial function assessments revealed a trend toward improvement in the treatment groups with respect to ejection fraction, viability, and cardiac index. However, histology of treated swine hearts identified no skeletal muscle cells. DISCUSSION: Percutaneous ASMT into an infarcted swine myocardium is feasible and safe, and may contribute to overall improved heart function.

Correlation of autologous skeletal myoblast survival with changes in left ventricular remodeling in dilated ischemic heart failure.

OBJECTIVES: The effect of autologous skeletal myoblast transplantation has not been rigorously studied in the setting of end-stage ischemic heart failure free of concomitant coronary revascularization. The aims of the present study were to determine autologous skeletal myoblast survival and its effects on left ventricular function and remodeling in sheep with dilated ischemic heart failure. METHODS: Ischemic heart failure (left ventricular ejection fraction, 30% +/- 2%; left ventricular end-systolic volume index, 82 +/- 9 mL/m2) was created in sheep (n = 11) with serial left circumflex coronary artery microembolizations. Instruments were inserted for the long-term determination of left ventricular global and regional dimensions, hemodynamics, and pressure-volume analysis after autologous skeletal myoblast transplantation (approximately 3.0 x 10(8) myoblasts; heart failure plus autologous skeletal myoblast group, n = 5) or without (heart failure-control group, n = 6). Measurements were performed in conscious animals. RESULTS: Autologous skeletal myoblast-derived skeletal muscle was found in all injected animals at 6 weeks. In ischemic heart failure, autologous skeletal myoblast cardiomyoplasty failed to improve systolic (left ventricular ejection fraction, 29% +/- 4%; dP/dT(max), 2863 +/- 152 mm Hg/s; end-systolic elastance, 1.6 +/- 0.22) or diastolic (left ventricular end-diastolic pressure, 21 +/- 2 mm Hg; time constant of relaxation (Tau), 34 +/- 4 ms; dP/dT(min), -1880 +/- 68 mm Hg/s) function. There was, however, attenuation in the left ventricular dilatation after autologous skeletal myoblast transplantation (change in end-systolic volume index, 14% +/- 4% vs 32% +/- 6%; P < .05). The effects of autologous skeletal myoblast-derived skeletal muscle were exclusive to the left ventricular short-axis dimension and dependent on autologous skeletal myoblast survival (R2 = 0.59, P = .006, n = 11). CONCLUSIONS: Autologous skeletal cardiomyoplasty was able to attenuate left ventricular remodeling in sheep with end-stage ischemic heart failure.

Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation.

OBJECTIVES: We report histological analysis of hearts from patients with end-stage heart disease who were transplanted with autologous skeletal myoblasts concurrent with left ventricular assist device (LVAD) implantation. BACKGROUND: Autologous skeletal myoblast transplantation is under investigation as a means to repair infarcted myocardium. To date, there is only indirect evidence to suggest survival of skeletal muscle in humans. METHODS: Five patients (all male; median age 60 years) with ischemic cardiomyopathy, refractory heart failure, and listed for heart transplantation underwent muscle biopsy from the quadriceps muscle. The muscle specimen was shipped to a cell isolation facility where myoblasts were isolated and grown. Patients received a transplant of 300 million cells concomitant with LVAD implantation. Four patients underwent LVAD explant after 68, 91, 141, and 191 days of LVAD support (three transplant, one LVAD death), respectively. One patient remains alive on LVAD support awaiting heart transplantation. RESULTS: Skeletal muscle cell survival and differentiation into mature myofibers were directly demonstrated in scarred myocardium from three of the four explanted hearts using an antibody against skeletal muscle-specific myosin heavy chain. An increase in small vessel formation was observed in one of three patients at the site of surviving myotubes, but not in adjacent tissue devoid of engrafted cells. CONCLUSIONS: These findings represent demonstration of autologous myoblast cell survival in human heart. The implanted skeletal myoblasts formed viable grafts in heavily scarred human myocardial tissue. These results establish the feasibility of myoblast transplants for myocardial repair in humans.