Raman microspectroscopic and dynamic vapor sorption characterization of hydration in collagen and dermal tissue

Water is an integral part of collagen's triple helical and higher order structure. Studies of model triple helical peptides have revealed the presence of repetitive intrachain, interchain, and intermolecular water bridges (Bella et al., Structure 1995, 15, 893-906). In addition, an extended cylinder of hydration is thought to be responsible for collagen fiber assembly. Confocal Raman spectroscopy and dynamic vapor sorption (DVS) measurements of human Type I collagen and pigskin dermis were performed to probe relative humidity (RH)-dependent differences in the nature and level of collagen hydration. Raman spectra were also acquired as a function of time for both Type I collagen and pigskin dermis samples upon exchange of a 100% RH H(2) O to deuterium oxide (D(2) O) environment. Alterations in Amide I and III modes were consistent with anticipated changes in hydrogen bonding strength as RH increased and upon H → D exchange. Of note is the identification of a Raman spectral marker (band at 938 cm(-1) ) which appears to be sensitive to alterations in collagen-bound water. Analysis of DVS isotherms provided a quantitative measure of adsorbed and absorbed water vapor consistent with the Raman results. The development of a Raman spectral marker of collagen hydration in intact tissue is relevant to diverse fields of study ranging from the evaluation of therapeutics for wound healing to hydration of aging skin.     

Ultrastructural and functional development of macrophages in the dermal tissue of rat fetuses

Summary After about 12 days of gestation, fetal macrophages begin to appear in the subepidermal mesenchyme of rat fetuses. The macrophages are ultrastructurally characterized by cytoplasmic vacuoles, abundant polyribosomes and long filopodia. Immunocytologically, they possess Fc and complement (C3) receptors on the cell surface and are capable of immune phagocytosis, Latex or carbon phagocytosis, and glass adherence. From 15 days of gestation, lysosomal granules and miropinocytic vesicles gradually develop, together with an enlargement of Golgi complexes, whereas the number of polysomes and the number and size of cytoplasmic vacuoles are gradually reduced when gestation ends. Finally, the macrophages become amoeboid. Non-specific esterase and endogenous peroxidase activities are always absent in these macrophages. In culture experiments with cell suspensions prepared from the mesenchyme, fetal macrophages show a similar maturation process. Autoradiography with ³H-thymidine demonstrates a high proliferative capacity of the macrophages, particularly during the fetal stage.Supported by Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan (No. 401057)     

The in vitro growth of a three-dimensional human dermal replacement using a single-pass perfusion system

A human dermal replacement has been developed by seeding human neonatal dermal fibroblasts onto a biosorbable polyglactin (polyglycolide/polylactide) mesh and culturing in a bioreactor. The mesh provides the proper environment for the cells to attach, grow in a three-dimensional array, and establish a tissue matrix over a 2- to 3-week culture period. The dermal replacement has been characterized and found to contain a variety of naturally occurring dermal matrix proteins, including fibronectin, glycosaminoglycans, and collagen types I and III. To efficiently and reproducibly produce this dermal tissue equivalent, a closed, single-pass perfusion system was developed and compared with a static process. In the single-pas perfusion system, growth medium (containing ascorbic acid) was perfused around the 4 x 6 in. pieces of mesh at specific flow rates determined by nutrient consumption and waste production rates. The flow rates used for this system indicate that a diffusion-limited regime exists with a mean residence time greater than 1 h for essential nutrients and factors. By controlling glucose concentrations in the system to a delta of 0.70 g/L from the inlet to the outlet of the bioreactor, it took 6 fewer days to grow a tissue similar to that produced by the static system.     

Three-dimensional epidermis-like growth of human mesenchymal stem cells on dermal equivalents: contribution to tissue organization by adaptation of myofibroblastic phenotype and function

Abstract Human mesenchymal stem cells (hMSC) are able to differentiate into mature cells of various mesenchymal tissues. Recent studies have reported that hMSC may even give rise to cells of ectodermal origin. This indication of plasticity makes hMSC a promising donor source for cell-based therapies. This study explores the differentiation potential of hMSC in a tissue-specific microenvironment simulated in vitro . HMSC were cultured air-exposed on dermal equivalents (DEs) consisting of collagen types I and III with dermal fibroblasts and subjected to conditions similar to those used for tissue engineering of skin with keratinocytes. Culture conditions were additionally modified by pre-treating the cells with 5-azacytidine or supplementing the medium with all trans retinoic acid (RA). HMSC were capable of adaptation to epidermis-specific conditions without losing their mesenchymal multipotency. However, despite the viability and evident three-dimensional epidermis-like growth pattern, hMSC showed a persistent expression of mesenchymal but not of epithelial markers, thus indicating a lack of epidermal (trans) differentiation. Further, electron microscopy and immunohistochemical analyses demonstrated that hMSC cultured under epidermis-specific conditions adopted a myofibroblastic phenotype and function, promoted in particular by air exposure. In conclusion, multipotent hMSC failed to differentiate into E-cadherin- or cytokeratin-expressing cells under optimized organotypic culture conditions for keratinocytes but differentiated into myofibroblast-like cells contracting the extracellular matrix, a phenomenon that was enhanced by RA and 5-azacytidine. These results indicate that hMSC might contribute to wound-healing processes by extracellular matrix reorganization and wound contraction but not by differentiation into keratinocytes.     

Differentiation capacity of stromal fibroblast-like cells from human bone marrow, adipose tissue, hair follicle dermal papilla and derma

We compared the morphology and differentiation capacity of human stromal cells derived from bone marrow (BMSC), adipose tissue (ATSC), hair follicle dermal papilla (DPC) and dermal fibroblasts (DFb). All cells have fibroblast-like morphology. ATSC and DPC cells expressed stem cell the surface markers CD105, CD49d, and STRO-1, which were revealed immunocytochemically. CD49d was not found on BMSC. The low expression of CD49d and STRO-1 was registered in the DFb population. ATSC, BMSC, and DPC have similar capacities for adipo- and osteogenic differentiation. These cells, cultured in appropriate induction media, alter the phenotype and synthesize specific proteins. However, the expression of differentiation in the DPC population is lower than in ATSC and BMSC cultures. We propose that these cell populations have primitive progenitor cells with properties of mesenchymal stem cells.     

Skin aging: Dermal adipocytes metabolically reprogram dermal fibroblasts

Emerging data connects the aging process in dermal fibroblasts with metabolic reprogramming, provided by enhanced fatty acid oxidation and reduced glycolysis. This switch may be caused by a significant expansion of the dermal white adipose tissue (dWAT) layer in aged, hair-covered skin. Dermal adipocytes cycle through de-differentiation and re-differentiation. As a result, there is a strongly enhanced release of free fatty acids into the extracellular space during the de-differentiation of dermal adipocytes in the catagen phase of the hair follicle cycle. Both caveolin-1 and adiponectin are critical factors influencing these processes. Controlling the expression levels of these two factors also offers the ability to manipulate the metabolic preferences of the different cell types within the microenvironment of the skin, including dermal fibroblasts. Differential expression of adiponectin and caveolin-1 in the various cell types may also be responsible for the cellular metabolic heterogeneity within the cells of the skin.     

The role of human immortal skin keratinocytes-acellular dermal matrix scaffold in skin repair and regeneration

In this study, we aimed to investigate the phenotypic characteristics of human immortal skin keratinocytes (HaCaT) cells and the role of acellular dermal matrix (ADM) in coculture system of HaCaT cells and ADM. Flow cytometry was used to examine the cluster of differentiation (CD) makers of HaCaT cells. Apoptosis analysis was applied to detect the apoptosis rate of HaCaT cells. Morphological observation of ADM isolated from the reticular layer of Sprague-Dawley rat dermis was utilized to evaluate the morphological structure of ADM. Methylthiazolyl tetrazolium (MTT) assay and morphological experiments were further used to confirm the scaffold role of ADM in HaCaT cells. A wound-healing mice model accompanied by HaCaT-ADM scaffold transplantation was performed to further verify the function of HaCaT-ADM scaffold. Our results showed that CD71, CD49f, K19, and CD29 were highly expressed in HaCaT cells, and the percentage of apoptosis cells was significantly increased, which represented that HaCaT cells had much stronger capacities of adhesion and proliferation than normal human keratinocytes. Additionally, the morphological structure of ADM presented many natural microbores, which made cells rapidly grow on ADM. The results exhibited that the HaCaT cells indeed promptly proliferate on ADM and easily grow into the microbores of ADM. Finally, an in vivo experiment further confirmed that the transplantation of the HaCaT-ADM scaffold into the dorsal skin of a wound-healing mice model could gradually repair the injured wound. Thus, these findings indicated that HaCaT cells might be as seed cells to develop skin tissue engineering and the HaCaT-ADM scaffold might be a better candidate to promote skin repair and regeneration.     

Compression-induced damage and internal tissue strains are related

Prolonged mechanical loading of soft tissues adjacent to bony prominences can lead to degeneration of muscle tissue, resulting in a condition termed pressure-related deep tissue injury. This type of deep pressure ulcers can develop into a severe wound, associated with problematic healing and a variable prognosis. Limited knowledge of the underlying damage pathways impedes effective preventive strategies and early detection. Traditionally, pressure-induced ischaemia has been thought to be the main aetiological factor for initiating damage. Recent research, however, proposes tissue deformation per se as another candidate for initiating pressure-induced deep tissue injury. In this study, different strain parameters were evaluated on their suitability as a generic predictive indicator for deep tissue injury. With a combined animal-experimental numerical approach, we show that there is a reproducible monotonic increase in damage with increasing maximum shear strain once a strain threshold has been exceeded. This relationship between maximum shear strain and damage seems to reflect an intrinsic muscle property, as it applied across a considerable number of the experiments. This finding confirms that tissue deformation per se is important in the aetiology of deep tissue injury. Using dedicated finite element modeling, a considerable reduction in the inherent biological variation was obtained, leading to the proposal that muscle deformation can prove a generic predictive indicator of damage.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.     

Osteochondral tissue coculture: An in vitro and in silico approach

Osteochondral tissue engineering aims to regenerate functional tissue-mimicking physiological properties of injured cartilage and its subchondral bone. Given the distinct structural and biochemical difference between bone and cartilage, bilayered scaffolds, and bioreactors are commonly employed. We present an osteochondral culture system which cocultured ATDC5 and MC3T3-E1 cells on an additive manufactured bilayered scaffold in a dual-chamber perfusion bioreactor. Also, finite element models (FEM) based on the microcomputed tomography image of the manufactured scaffold as well as on the computer-aided design (CAD) were constructed; the microenvironment inside the two FEM was studied and compared. In vitro results showed that the coculture system supported osteochondral tissue growth in terms of cell viability, proliferation, distribution, and attachment. In silico results showed that the CAD and the actual manufactured scaffold had significant differences in the flow velocity, differentiation media mixing in the bioreactor and fluid-induced shear stress experienced by the cells. This system was shown to have the desired microenvironment for osteochondral tissue engineering and it can potentially be used as an inexpensive tool for testing newly developed pharmaceutical products for osteochondral defects.     

A novel micro-to-macro approach for cardiac tissue mechanics

For studying cardiac mechanics, hyperelastic anisotropic computational models have been developed which require the tissue anisotropic and hyperelastic parameters. These parameters are obtained by tissue samples mechanically testing. The validity of such parameters are limited to the specific tissue sample only. They are not adaptable for pathological tissues commonly associated with tissue microstructure alterations. To investigate cardiac tissue mechanics, a novel approach is proposed to model hyperelasticity and anisotropy. This approach is adaptable to various tissue microstructural constituent’s distributions in normal and pathological tissues. In this approach, the tissue is idealized as composite material consisting of cardiomyocytes distributed in extracellular matrix (ECM). The major myocardial tissue constituents are mitochondria and myofibrils while the main ECM’s constituents are collagen fibers and fibroblasts. Accordingly, finite element simulations of uniaxial and equibiaxial tests of normal and infarcted tissue samples with known amounts of these constituents were conducted, leading to corresponding tissue stress–strain data that were fitted to anisotropic/hyperelastic models. The models were validated where they showed good agreement characterized by maximum average stress-strain errors of 16.17 and 10.01% for normal and infarcted cardiac tissue, respectively. This demonstrate the effectiveness of the proposed models in accurate characterization of healthy and pathological cardiac tissues.     

Influence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents: A box-behnken analysis

Summary Our laboratory has been involved in finding optimal conditions for producing dermal and skin equivalents. As an original approach, a Box-Behnken experimental design was used to study the effects of the initial collagen and fibroblast concentrations and the initial gel thickness on the contraction of dermal and skin equivalents. The final surface area of dermal equivalent varied significantly with the initial concentration of collagen and fibroblast, whereas the initial thickness of gel had no appreciable effect on the contraction of the dermal equivalent. When keratinocytes were grown on these dermal equivalents they produced a very severe contraction, to an extent that all skin equivalents had a similar final surface area. This severe contraction was independent of collagen and fibroblast concentrations. Models for the prediction of the final percentage contraction of dermal and skin equivalents as a function of the initial concentration of collagen, the logarithm of fibroblast concentration, and the initial gel thickness were obtained and analyzed. Keratinocytes grown at the lowest seeding density did not contract the equivalents. However, histologic analysis has shown an incomplete coverage by these cells of the equivalents. The extensive contraction of the skin equivalent presenting adequate morphology is a major drawback toward its clinical utilization for burn wound coverage. The financial supports for this project were received from Canadian NSERC postgraduate scholarship (P. Rompré), Québec FCAR postgraduate scholarship (C.A. López Valle), France-Québec research grant in Biotechnology (F.A. Auger), Canadian MRC grant (F.A. Auger), and NSERC grants (A. LeDuy and J. Thibault).     

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.     

Embryonic wound healing: A primer for engineering novel therapies for tissue repair

Scar is the default tissue repair used by the body in response to most injuries–a response that occurs in wounds ranging in seriousness from minor skin cuts to complete severance of the spinal cord. By contrast, before the third trimester of pregnancy embryonic mammals tend to heal without scarring due to a variety of mechanisms and factors that are uniquely in operation during development in utero. The goal of tissue engineering is to develop safe and clinically effective biological substitutes that restore, maintain, or improve tissue function in patients. This review provides a comparative overview of wound healing during development and maturation and seeks to provide a perspective on just how much the embryo may be able teach us in the engineering of new therapies for tissue repair. Birth Defects Research (Part C) 96:258–270, 2012. © 2012 Wiley Periodicals, Inc.