Extracellular matrix in skin diseases: The road to new therapies.

BACKGROUND: The extracellular matrix (ECM) is a vital structure with a dynamic and complex organization that plays an essential role in tissue homeostasis. In the skin, the ECM is arranged into two types of compartments: interstitial dermal matrix and basement membrane (BM). All evidence in the literature supports the notion that direct dysregulation of the composition, abundance or structure of one of these types of ECM, or indirect modifications in proteins that interact with them is linked to a wide range of human skin pathologies, including hereditary, autoimmune, and neoplastic diseases. Even though the ECM's key role in these pathologies has been widely documented, its potential as a therapeutic target has been overlooked. AIM OF REVIEW: This review discusses the molecular mechanisms involved in three groups of skin ECM-related diseases - genetic, autoimmune, and neoplastic - and the recent therapeutic progress and opportunities targeting ECM. KEY SCIENTIFIC CONCEPTS OF REVIEW: This article describes the implications of alterations in ECM components and in BM-associated molecules that are determinant for guaranteeing its function in different skin disorders. Also, ongoing clinical trials on ECM-targeted therapies are discussed together with future opportunities that may open new avenues for treating ECM-associated skin diseases.

Mechanical Property of Hydrogels and the Presence of Adipose Stem Cells in Tumor Stroma Affect Spheroid Formation in the 3D Osteosarcoma Model.

Osteosarcoma is one of the most common metastatic bone cancers, which results in significant morbidity and mortality. Unfolding of effectual therapeutic strategies against osteosarcoma is impeded because of the absence of adequate animal models, which can truly recapitulate disease biology of humans. Tissue engineering provides an opportunity to develop physiologically relevant, reproducible, and tunable in vitro platforms to investigate the interactions of osteosarcoma cells with its microenvironment. Adipose-derived stem cells (ASCs) are detected adjacent to osteosarcoma masses and are considered to have protumor effects. Hence, the present study focuses on investigating the role of reactive ASCs in formation of spheroids of osteosarcoma cells (Saos 2) within a three-dimensional (3D) niche, which is created using gellan gum (GG)-silk fibroin. By modifying the blending ratio of GG-silk, the optimum stiffness of the resultant hydrogels such as GG and GG75: S25 is obtained for cancer spheroid formation. This work indicates that the co-existence of cancer and stem cells can form a spheroid, the hallmark of cancer, only in particular microenvironment stiffness. The incorporation of fibrillar silk fibroin within the hydrophilic network of GG in GG75: S25 spongy-like hydrogels closely mimics the stiffness of commercially established cancer biomaterials (e.g., Matrigel, HyStem). The GG75: S25 hydrogel maintains the metabolically active construct for a longer time with elevated expression of osteopontin, osteocalcin, RUNX 2, and bone sialoprotein genes, the biomarkers of osteosarcoma, compared to GG. The GG75: S25 construct also exhibits intense alkaline phosphatase expression in immunohistochemistry compared to GG, indicating itspotentiality to serve as biomimetic niche to model osteosarcoma. Taken together, the GG-silk fibroin-blended spongy-like hydrogel is envisioned as an alternative low-cost platform for 3D cancer modeling.

Cell selective chitosan microparticles as injectable cell carriers for tissue regeneration.

The detection, isolation and sorting of cells holds an important role in cell therapy and regenerative medicine. Also, injectable systems have been explored for tissue regeneration in vivo, because it allows repairing complex shaped tissue defects through minimally invasive surgical procedures. Here we report the development of chitosan microparticles with a size of 115.8 µm able to capture and expand a specific cell type that can also be regarded as an injectable biomaterial. Monoclonal antibodies against cell surface antigens specific to endothelial cells and stem cells were immobilized on the surface of the microparticles. Experimental results showed that particles bioconjugated with specific antibodies provide suitable surfaces to capture a target cell type and subsequent expansion of the captured cells. Primarily designed for an application in tissue engineering, three main challenges are accomplished with the herein presented microparticles: separation, scale-up expansion of specific cell type and successful use as an injectable system to form small tissue constructs in situ.

Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds.

Skin regeneration remains a challenge, requiring a well-orchestrated interplay of cell-cell and cell-matrix signalling. Cell sheet (CS) engineering, which has the major advantage of allowing the retrieval of the intact cell layers along with their naturally organized extracellular matrix (ECM), has been poorly explored for the purpose of creating skin substitutes and skin regeneration. This work proposes the use of CS technology to engineer cellular constructs based on human keratinocytes (hKC), key players in wound re-epithelialization, dermal fibroblasts (hDFb), responsible for ECM remodelling, and dermal microvascular endothelial cells (hDMEC), part of the dermal vascular network and modulators of angiogenesis. Homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed simultaneously to target wound re-vascularization and re-epithelialization. After implantation of the constructs in murine full-thickness wounds, human cells were engrafted into the host wound bed and were present in the neotissue formed up to 14 days post-implantation. Different outcomes were obtained by varying the composition and organization of the 3-D constructs. Both hKC and hDMEC significantly contributed to re-epithelialization by promoting rapid wound closure and early epithelial coverage. Moreover, a significant increase in the density of vessels at day 7 and the incorporation of hDMEC in the neoformed vasculature confirmed its role over neotissue vacularization. As a whole, the obtained results confirmed that the proposed 3-D CS-based constructs provided the necessary cell machinery, when in a specific microenvironment, guiding both re-vascularization and re-epithelialization. Although dependent on the nature of the constructs, the results obtained sustain the hypothesis that different CS-based constructs lead to improved skin healing.

Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds.

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14.

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

Changes in lipid and lipoprotein levels and body weight in Tarahumara Indians after consumption of an affluent diet.

BACKGROUND: Major new public health problems occur in developing countries as they become more affluent and change their traditional dietary patterns. To study this phenomenon in microcosm, we substituted an "affluent" diet for the traditional diet of a group of Tarahumara Indians, a Mexican people known to consume a low-fat, high-fiber diet and to have a very low incidence of risk factors for coronary heart disease. METHODS: Thirteen Tarahumara Indians (five women and eight men [including one adolescent]) consumed their traditional diet (2700 kcal per day) for one week, and were then fed a diet typical of affluent societies, which contained excessive calories (4100 kcal per day), total fat, saturated fat, and cholesterol, for five weeks. RESULTS: After five weeks of consuming the affluent diet, the subjects' mean (+/- SE) plasma cholesterol level increased by 31 percent, from 121 +/- 5 to 159 +/- 6 mg per deciliter (3.13 +/- 0.13 to 4.11 +/- 0.16 mmol per liter, P less than 0.001). The increase in the plasma cholesterol level was primarily in the low-density lipoprotein (LDL) fraction, which rose 39 percent, from 72 +/- 3 to 100 +/- 4 mg per deciliter (1.86 +/- 0.08 to 2.59 +/- 0.10 mmol per liter, P less than 0.001). High-density lipoprotein (HDL) cholesterol, usually low in this population, increased by 31 percent, from 32 +/- 2 to 42 +/- 3 mg per deciliter (0.83 +/- 0.05 to 1.09 +/- 0.08 mmol per liter). Consequently, the ratio of LDL to HDL levels changed little (2.25 with the base-line diet and 2.38 with the affluent diet). Plasma triglyceride levels increased by 18 percent, from 91 +/- 8 to 108 +/- 11 mg per deciliter (1.03 +/- 0.09 to 1.22 +/- 0.12 mmol per liter, P less than 0.05), with a significant increase in the very-low-density lipoprotein triglyceride fraction. All the subjects gained weight, with a mean increase of 3.8 kg (7 percent). CONCLUSIONS: When Tarahumara Indians from a population with virtually no coronary risk factors consumed for a short time a hypercaloric diet typical of a more affluent society, they had dramatic increases in plasma lipid and lipoprotein levels and body weight. If sustained, such changes might increase their risk of coronary heart disease.

The absorption of cholesterol and the sterol balance in the Tarahumara Indians of Mexico fed cholesterol-free and high cholesterol diets.

The Tarahumara Indians of Mexico are habituated to a very low cholesterol, low fat diet and have lifelong low plasma cholesterol concentrations. To study cholesterol metabolism in these unusual people, 8 Tarahumara men were fed sequentially a cholesterol-free diet and then a diet containing 900 mg cholesterol under controlled conditions. The intestinal absorption of cholesterol, fecal steroid excretion and sterol balance were determined. During the high cholesterol diet period, the plasma cholesterol level increased from 113 +/- 8 mg/dl to 147 +/- 11 mg/dl (means +/- SD). Cholesterol biosynthesis decreased from 14.0 +/- 0.7 to 7.1 +/- 1.0 mg/kg/day (means +/- SE). The intestinal absorption of cholesterol was 27.7 +/- 6.7% (means +/- SE) during both dietary periods. Compared to other cultures, Tarahumaras had a reduced ability to absorb dietary cholesterol and higher total sterol turnover primarily because of an increased bile acid output. The total sterol disposition over three weeks of the high cholesterol diet accounted for all the absorbed dietary cholesterol.

Dietary cholesterol and the plasma lipids and lipoproteins in the Tarahumara Indians: a people habituated to a low cholesterol diet after weaning.

Eight Tarahumara Indian men participated in a metabolic study to measure the responsiveness of their plasma cholesterol levels to dietary cholesterol. They were fed isocaloric cholesterol-free and high cholesterol diets containing 20% fat, 15% protein, and 65% carbohydrate calories. On admission to the study, the Tarahumaras had a low mean plasma cholesterol concentration (120 mg/dl), reflecting their habitual low cholesterol diet. After 3 wk of a cholesterol-free diet their cholesterol levels were 113 mg/dl. The men were then fed a high cholesterol diet (1000 mg/day) which increased the mean total plasma cholesterol to 147 mg/dl (p less than 0.01) and also increased the low-density lipoprotein cholesterol concentration. Tarahumaras, habituated to a low cholesterol diet after weaning, had the typical hypercholesterolemic response to a high cholesterol diet that has been previously observed in subjects whose lifelong diet was high in cholesterol content.

The food and nutrient intakes of the Tarahumara Indians of Mexico.

A nutritional survey of 372 semiacculturated Tarahumara Indians in the Sierra Madre Occidental Mountains of Mexico was carried out to determine the composition of their diet and its nutritional adequacy. Dietary histories from 174 adults and 198 children were obtained by interviews and field observations during 1973 and 1974. The histories for the children were calculated in part from the menus of six boarding church schools. Nutrient calculations of daily intake were based upon food composition tables and some actual analyses of Tarahumara foods. The protein intake was ample, at 87 g, and generously met the FAO/WHO recommendations for daily intake of essential amino acids. Fat contributed only 12% of total calories, its composition being 2% saturated and 5% polyunsaturated with a P/S ratio of 2. The mean dietary cholesterol intake was very low, less than 100 mg/day, and the plant sterol intake was high, over 400 mg/day. Carbohydrate comprised 75 to 80% of total calories, mostly from starch. Only 6% of total calories were derived from simple sugars. The crude fiber intake was high, 18 to 21 g/day. Salt consumption was moderately low, 5 to 8 g/day. The daily intakes of calcium, iron, vitamin A, ascorbic acid, thiamin niacin, riboflavin, and vitamin B6 exceeded or approximated the FAO/WHO recommendations. Thus, the simple diet of the Tarahumara Indians, composed primarily of beans and corn, provided a high intake of complex carbohydrate and was low in fat and cholesterol. Their diet was found to be generally of high nutritional quality and would, by all criteria, be considered antiatherogenic.