Dermal Fibroblasts as the Main Target for Skin Anti-Age Correction Using a Combination of Regenerative Medicine Methods.

This article includes the data from current studies regarding the pathophysiological mechanisms of skin aging and the regenerative processes occurring in the epidermis and dermis at the molecular and cellular level, mainly, the key role of dermal fibroblasts in skin regeneration. Analyzing these data, the authors proposed the concept of skin anti-age therapy that is based on the correction of age-related skin changes by stimulating regenerative processes at the molecular and cellular level. The main target of the skin anti-age therapy is dermal fibroblasts (DFs). A variant of the cosmetological anti-age program using the combination of laser and cellular methods of regenerative medicine is presented in the paper. The program includes three stages of implementation and defines the tasks and methods of each stage. Thus, laser technologies allow one to remodel the collagen matrix and create favorable conditions for DFs functions, whereas the cultivated autologous dermal fibroblasts replenish the pool of mature DFs decreasing with age and are responsible for the synthesis of components of the dermal extracellular matrix. Finally, the use of autological platelet-rich plasma (PRP) enables to maintenance of the achieved results by stimulating DF function. It has been shown that growth factors/cytokines contained in α-granules of platelets injected into the skin bind to the corresponding transmembrane receptors on the surface of DFs and stimulate their synthetic activity. Thus, the consecutive, step-by-step application of the described methods of regenerative medicine amplifies the effect on the molecular and cellular aging processes and thereby allows one to optimize and prolong the clinical results of skin rejuvenation.

Correlation of the regenerative potential of dermal fibroblasts in 2D culture with the biological properties of fibroblast-derived tissue spheroids.

In situ 3D bioprinting is a new emerging therapeutic modality for treating human skin diseases. The tissue spheroids have been previously suggested as a powerful tool in rapidly expanding bioprinting technology. It has been demonstrated that the regenerative potential of human dermal fibroblasts could be quantitatively evaluated in 2D cell culture and confirmed after implantation in vivo. However, the development of unbiassed quantitative criteria of the regenerative potential of 3D tissue spheroids in vitro before their in situ bioprinting remains to be investigated. Here it has been demonstrated for the first time that specific correlations exist between the regenerative potential of human dermal fibroblasts cultured in vitro as 2D cell monolayer with biological properties of 3D tissue spheroids fabricated from these fibroblasts. In vitro assessment of biological properties included diameter, spreading and fusion kinetics, and biomechanical properties of 3D tissue spheroids. This comprehensive characterization could be used to predict tissue spheroids' regenerative potential in vivo.

Age-Related Changes in the Fibroblastic Differon of the Dermis: Role in Skin Aging.

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. The processes developing in the skin during aging are based on fundamental molecular mechanisms associated with fibroblasts, the main cellular population of the dermis. It has been revealed that the amount of fibroblasts decreases markedly with age and their functional activity is also reduced. This inevitably leads to a decrease in the regenerative abilities of the skin and the progression of its aging. In this review we consider the mechanisms underlying these processes, mainly the changes observed with age in the stem/progenitor cells that constitute the fibroblastic differon of the dermis and form their microenvironment (niches). These changes lead to the depletion of stem cells, which, in turn, leads to a decrease in the number of differentiated (mature) dermal fibroblasts responsible for the production of the dermal extracellular matrix and its remodeling. We also describe in detail DNA damages, their cellular and systemic consequences, molecular mechanisms of DNA damage response, and also the role of fibroblast senescence in skin aging.

Molecular Mechanisms of Changes in Homeostasis of the Dermal Extracellular Matrix: Both Involutional and Mediated by Ultraviolet Radiation.

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. With age, an impairment of structures, quality characteristics, and functions of the dermal extracellular matrix (ECM) occurs in the skin, which leads to disrupted functioning of dermal fibroblasts (DFs), the main cells supporting morphofunctional organization of the skin. The DF functioning directly depends on the state of the surrounding collagen matrix (CM). The intact collagen matrix ensures proper adhesion and mechanical tension in DFs, which allows these cells to maintain collagen homeostasis while ECM correctly regulates cellular processes. When the integrity of CM is destroyed, mechanotransduction is disrupted, which is accompanied by impairment of DF functioning and destruction of collagen homeostasis, thereby contributing to the progression of aging processes in skin tissues. This article considers in detail the processes of skin aging and associated changes in the skin layers, as well as the mechanisms of these processes at the molecular level.

Spontaneous γH2AX foci in human dermal fibroblasts in relation to proliferation activity and aging.

We assessed the effects of donor age on clonogenicity, proliferative potential, and spontaneous γH2AX foci in the proliferating (Ki67 +) and senescent (SA ß-gal +) cultures of skin fibroblasts isolated from 34 donors of different age (23-82 years). Here, we demonstrated that neither the colony forming effectiveness of proliferating (Ki67+) fraction of the fibroblasts nor the average number of γH2AX foci of the same fraction does not depend on the age of the donor. The correlation between the number of γH2AX foci and the donor's age was reliable in quiescent (Ki67-) cells. The average number of γH2AX foci in quiescent fibroblasts of donors older than 68 years was about two times higher than in the same cells of up to 30 years old donors. The number of γH2AX foci demonstrated a statistically significant positive correlation with the fraction of proliferating cells in fibroblast cultures. On average, proliferating cells have twice as many the γH2AX foci in comparison with the quiescent cells. Within a population of proliferating (Ki67+) cells, the degree of senescence correlated with a relative declining of constitutive γH2AX foci number, whereas in the population of quiescent (Ki67-) cells, it was proportional to augmenting the number of the γH2AX foci. Our data on a statistically significant (p=0.001) correlation between the age of the donor and the number of constitutive γH2AX foci in quiescent cells, could point out the ongoing DNA-damage response due in the maintenance of the senescent state of cells.

Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation.

Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated ß-galactosidase (SA-ßgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-ßgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-ßgal positive cells in mass culture. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=-0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=-0.94, p=0.0001) between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

Myogenic potential of human alveolar mucosa derived cells.

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.

Clinical-instrumental and morphological evaluation of the effect of autologous dermal fibroblasts administration.

Basic molecular mechanisms, associated with the main cell population of the dermis - fibroblasts - are the basis of skin aging. The number of functionally active fibroblasts in the skin and their biosynthetic activity decreases with age, thus enhancement of their cell density with synthetically active cells is accepted as a one of the most effective methods. The objective of the present study was to evaluate the safety and effectiveness of intradermal administration of autologous dermal fibroblasts in a year after treatment of 17 patients, aged 45-65 years. Results obtained with modern instrumental skin diagnostic methods (vacuum cutometry, optical profilometry, VISIA photometric analysis, etc.) demonstrate the safety and clinical effectiveness of dermal autofibroblast therapy: after transplantation, cultured autofibroblasts keep their biosynthetic activity and produce extracellular matrix for at least 12 months. As a result, remodelling of the dermis microstructures is observed, accompanied by a progressive increase of collagen content and thickness of the dermis (up to 62.5 ±6.7% in 12 months). This is clinically expressed by increase of skin elasticity (24.0 ±4.3% in periorbital area) and thickness of the skin, and by decrease in the number and depth of wrinkles (46 ±7% by the end of observation period). Copyright © 2014 John Wiley & Sons, Ltd.

Octacalcium phosphate ceramics combined with gingiva-derived stromal cells for engineered functional bone grafts.

Biocompatible ceramic fillers are capable of sustaining bone formation in the proper environment. The major drawback of these scaffolding materials is the absence of osteoinductivity. To overcome this limitation, bioengineered scaffolds combine osteoconductive components (biomaterials) with osteogenic features such as cells and growth factors. The bone marrow mesenchymal stromal cells (BMMSCs) and the ß-tricalcium phosphate (ß-TCP) are well-known and characterized in this regard. The present study was conducted to compare the properties of novel octacalcium phosphate ceramic (OCP) granules with ß-TCP (Cerasorb(®)), gingiva-derived mesenchymal stromal cells (GMSCs) properties with the BMMSCs and osteogenic and angiogenic properties of a bioengineered composite based on OCP granules and the GMSCs. This study demonstrates that GMSCs and BMMSСs have a similar osteogenic capacity. The usage of OCP ceramic granules in combination with BMMSCs/GMSCs significantly affects the osteo- and angiogenesis in bone grafts of ectopic models.