Dose-dependent dominance: How cell densities design stromal cell functions during soft tissue healing.

Regular soft tissue healing relies on the well-organized interaction of different stromal cell types with endothelial cells. However, spatiotemporal conditions might provoke high densities of one special stromal cell type, potentially leading to impaired healing. Detailed knowledge of the functions of rivaling stromal cell types aiming for tissue contraction and stabilization as well as vascular support is mandatory. By the application of an in vitro approach comprising the evaluation of cell proliferation, cell morphology, myofibroblastoid differentiation, and cytokine release, we verified a density-dependent modulation of these functions among juvenile and adult fibroblasts, pericytes, and adipose-derived stem cells during their interaction with microvascular endothelial cells in cocultures. Results indicate that juvenile fibroblasts rather support angiogenesis via paracrine regulation at the early stage of healing, a role potentially compromised in adult fibroblasts. In contrast, pericytes showed a more versatile character aiming at angiogenesis, vessel stabilization, and tissue contraction. Such a universal character was even more pronounced among adipose-derived stem cells. The explicit knowledge of the characteristic functions of stromal cell types is a prerequisite for the development of new analytical and therapeutic approaches for impaired soft tissue healing. The present study delivers new considerations concerning the roles of rivaling stromal cell types within a granulation tissue, pointing to extraordinary properties of pericytes and adipose-derived stem cells.

Interacting adipose-derived stem cells and microvascular endothelial cells provide a beneficial milieu for soft tissue healing.

There is growing evidence suggesting that healing of chronic soft tissue wounds profits from the presence of adipose-derived stem cells (ADSC). Among the large spectrum of mechanisms by which ADSC might act, especially the interaction with the microvascular endothelial cell, a main player during angiogenesis, is of special interest. In the present 2D model on the basis of endothelial cell ADSC co-cultures, we focused on the identification of characteristics of both cell types in response to a typical condition in acute and chronic wounds: hypoxia. Parameters like proliferation capacity, migration, myofibroblastoid differentiation of ADSC and the quantification of important paracrine factors related to angiogenesis and inflammation were used to correlate our experimental model with the in vivo situation of soft tissue healing. ADSC were not negatively affected by hypoxia in terms of proliferation, referring to their excellent hypoxia tolerance. Myofibroblastoid differentiation among ADSC was enhanced by hypoxia in mono- but not in co-culture. Furthermore, co-cultures were able to migrate under hypoxia. These effects might be caused to some extent by the distinct milieu created by interacting ADSC and endothelial cells, which was characterized by modulated levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor. The identification of these cell characteristics in the present 2D in vitro model provide new insights into the process of human soft tissue healing, and underpin a beneficial role of ADSC by regulating inflammation and angiogenesis.

Controlling fibroblast adhesion and proliferation by 1D Al2O3 nanostructures.

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast-surface interaction. In the present study, one-dimensional aluminium oxide (1D Al2O3) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well-defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al2O3 nanostructures on the substratum.

The role of adipose-derived stem cells in a self-organizing 3D model with regard to human soft tissue healing.

The clinical phenomenon of inadequate soft tissue healing still remains an important issue. The occurrence of chronic wounds is correlated to the life span, which is still increasing in western countries. Tissue engineering products containing adipose-derived stem cells are discussed as a promising therapeutic approach. Several studies confirmed the value of these cells for soft tissue healing improvement, suggesting a paracrine as well as a direct effect on vessel repair and angiogenesis. In an attempt to figure out specific effects of adipose-derived stem cells on dermal microvascular endothelial cells with respect to the different phases of soft tissue healing, we designed a 3D in vitro model on the basis of spheroids. Basic parameters like spheroid volume, cell numbers, and rate of apoptotic cells were determined in dependence on culture time, on different oxygen conditions and using mono- as well as co-cultures of both cell types. Furthermore we focused on gene expression and protein levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor, which are discussed against the background of therapies for chronic wounds. The visualization of α-smooth muscle actin allowed the estimation of the function of adipose-derived stem cells as stabilizer for dermal microvascular endothelial cells. The results of the present 3D model underscore a paracrine effect of adipose-derived stem cells on microvessel repair during early hypoxic conditions, whereas a stabilizing effect occurs during a later phase of soft tissue healing, simultaneously to reoxygenation.

Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis.

BACKGROUND: Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. METHODS: We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy®-technology and the cellular composition was analyzed by flow cytometry. RESULTS: Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase®, whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. DISCUSSION: Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size.

Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction.

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.

Response of endothelial cells and pericytes to hypoxia and erythropoietin in a co-culture assay dedicated to soft tissue repair.

The increasing mean life expectancy of the citizens of the western world countries leads to an increase of the age-related diseases, among them soft tissue defects exhibiting inadequate healing. In order to develop new therapeutic strategies to support disturbed soft tissue repair, there is a strong need of sophisticated in vitro assays. A new assay combining scratch wounding with co-cultures of primary human microvascular endothelial cells (HDMEC) and pericytes (HPC) focuses on basic characteristics of cell interaction against the background of soft tissue repair. The cell parameters proliferation, migration and differentiation, and the release of monocyte chemoattractant protein-1 (MCP-1) were analysed in response to hypoxia (pO2 < 5 mmHg) and to erythropoietin (EPO; 50 IU/ml), a glycoprotein hormone having shown promising effects in soft tissue repair. As basic characteristics of the assay, direct cell contact in co-culture led to a weakened proliferation of both cell types, an increase of the percentage of myofibroblast-like pericytes and to a higher release of MCP-1. Hypoxia caused a proliferation decrease of HPC in co-culture, which was slightly attenuated by EPO. Hypoxia also reduced the MCP-1 release of co-cultured cells, when EPO had been added. In addition, EPO had a rather positive effect on HPC migration under hypoxia. These in vitro results allow new insights into the interaction of pericytes with endothelial cells in the context of soft tissue repair.

Analysis of cellular composition of co-culture spheroids.

3D spheroids and in particular co-culture spheroids reflect the natural organization of cells in tissues much better than 2D cell cultures as indicated by differences in cellular phyisology. However, most methods to analyze cells were established for 2D cultures and cannot easily be applied to spheroids. This study has aimed to demonstrate the possibility of quantification of the cellular composition of co-culture spheroids without previous dissociation into single cells. Prior to the generation of the spheroids, human endothelial cells, osteoblasts and fibroblasts were stained with fluoresent dyes for living cells. Co-culture spheroids of defined stoichiometric compositions were generated by the liquid overlay technique, cultivated for one, three or six days, respectively, and afterwards snap-frozen in liquid nitrogen. Cryo-sections of co-culture spheroids were analyzed by fluorescence microscopy and a newly established semi-automatic measuring routine. In order to compare the results, spheroids of one group were dissociated and the cellular composition was quantified by FACS-analysis. Staining efficiencies were higher than 95% as quantified in preliminary experiments with 2D cultures. Depending on the staining procedure, variations from uniform to punctate signals were detected. The size of all co-culture spheroids decreased over time and snap-freezing did not lead to shrinkage of the spheroids. We were able to detect organizational patterns of different cell types within the spheroids. It was possible to determine the cellular composition by quantitative microscopic analyses of cryo-sections as it could be confirmed by flow cytometric analyses. Depending on the experimental requirements, a combination of both methods might lead to valuable synergy.

Induction of osteogenic differentiation by nanostructured alumina surfaces.

Permanent orthopedic implants are becoming increasingly important due to the demographic development. Their optimal osseointegration is key in obtaining good secondary stability. For anchorage dependent cells, topographic features of a surface play an essential role for cell adhesion, proliferation, differentiation and biomineralization. We studied the topographical effect of nanostructured alumina surfaces prepared by chemical vapor deposition on osteogenic differentiation and growth of human osteoblasts. Chemical vapor deposition of the single source precursor (tBuOAIH2)2 led to synthesis of one dimensional alumina nanostructures of high purity with a controlled stoichiometry. We fabricated different topographic features by altering the distribution density of deposited one dimensional nanostructures. Although the topography differed, all surfaces exhibited identical surface chemistry, which is the key requirement for systematically studying the effect of the topography on cells. Forty-eight hours after seeding, cell density and cell area were not affected by the nanotopography, whereas metabolic activity was reduced and formation of actin-fibres and focal adhesions was impaired compared to the uncoated control. Induction of osteogenic differentiation was demonstrated via up-regulation of alkaline phosphatase, bone sialoprotein, osteopontin and Runx2 at the mRNA level, demonstrating the potential of nanostructured surfaces to improve the osseointegration of permanent implants.

Bromelain down-regulates myofibroblast differentiation in an in vitro wound healing assay.

Bromelain, a pineapple-derived enzyme mixture, is a widely used drug to improve tissue regeneration. Clinical and experimental data indicate a better outcome of soft tissue healing under the influence of bromelain. Proteolytic, anti-bacterial, anti-inflammatory, and anti-oedematogenic effects account for this improvement on the systemic level. It remains unknown, whether involved tissue cells are directly influenced by bromelain. In order to gain more insight into those mechanisms by which bromelain modulates tissue regeneration at the cellular level, we applied a well-established in vitro wound healing assay. Two main players of soft tissue healing--fibroblasts and microvascular endothelial cells--were used as mono- and co-cultures. Cell migration, proliferation, apoptosis, and the differentiation of fibroblasts to myofibroblasts as well as interleukin-6 were quantified in response to bromelain (36 × 10(-3) IU/ml) under normoxia and hypoxia. Bromelain attenuated endothelial cell and fibroblast proliferation in a moderate way. This proliferation decrease was not caused by apoptosis, rather, by driving cells into the resting state G0 of the cell cycle. Endothelial cell migration was not influenced by bromelain, whereas fibroblast migration was clearly slowed down, especially under hypoxia. Bromelain led to a significant decrease of myofibroblasts under both normoxic (from 19 to 12 %) and hypoxic conditions (from 22 to 15 %), coincident with higher levels of interleukin-6. Myofibroblast differentiation, a clear sign of fibrotic development, can be attenuated by the application of bromelain in vitro. Usage of bromelain as a therapeutic drug for chronic human wounds thus remains a very promising concept for the future.

Soft tissue fibroblasts from well healing and chronic human wounds show different rates of myofibroblasts in vitro.

Due to an increasing life expectancy in western countries, chronic wound treatment will be an emerging challenge in the next decades. Because therapies are improving slowly appropriate diagnostic tools enabling the early prediction of the healing success remain to be developed. We used a well-established in vitro assay in combination with the analysis of 27 cytokines to discriminate between fibroblasts from chronic (n = 6) and well healing (n = 8) human wounds. Proliferation and migration of the cells as well as their response to hypoxia and their behaviour in co-culture with microvascular endothelial cells were analyzed. Myofibroblast differentiation, a time-limited essential process of regular wound healing, was also quantified. Besides weaker proliferation and migration significantly higher rates of myofibroblasts were detected in chronic wounds. With respect to the cytokine release, there was a clear trend within the group of chronic wound fibroblasts, which were releasing interferon-γ, monocyte chemotactic protein-1, granulocyte-macrophage colony stimulating factor and basic fibroblast growth factor in higher amounts than fibroblasts from healing wounds. Although the overall response of both groups of fibroblasts to hypoxia and to the contact with endothelial cells was similar, especially chronic wound fibroblasts seemed to benefit from the endothelial interaction during hypoxia and displayed better migration characteristics. The study shows (1) that the assay can identify specific features of fibroblasts derived from different human wounds and (2) that wound fibroblasts are varying in their response to the chosen parameters. Thus, current therapeutic approaches and individual healing prediction might benefit from this assay.

Reduced myofibroblast differentiation on femtosecond laser treated 316LS stainless steel.

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316 LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 µm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

BACKGROUND AIMS: The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. METHODS: We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. RESULTS: The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. CONCLUSIONS: Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

Erythropoietin ameliorates the reduced migration of human fibroblasts during in vitro hypoxia

No disponible

Erythropoietin promotes the formation of granulation tissue when administered to soft tissue wounds and it was shown to be most effective under tissue hypoxia. However, the action of erythropoietin on the cellular level is not well understood. In order to get a better insight into these processes, an in vitro wound healing assay was applied. Two main players of soft tissue healing—fibroblasts and microvascular endothelial cells—were used as mono- and co-cultures, subsequently inflicting in vitro wounds. Cell migration, proliferation, the differentiation of fibroblasts to myofibroblasts, and the release of vascular endothelial cell growth factor A and angiogenin were quantified in response to hypoxia and erythropoietin (5 IU/ml). Erythropoietin supplementation did neither affect proliferation nor migration of endothelial cells and fibroblasts under normoxia. Under hypoxia, the reduced fibroblast migration was ameliorated by erythropoietin. This effect coincided with an attenuated release of vascular endothelial growth factor A, whereas angiogenin release was unaffected by erythropoietin. The in vitro model applied in this study may represent an adequate approximation to certain aspects of the in vivo status of soft tissue regeneration and the results might serve to interpret the in vivo efficiency of erythropoietin at the cellular level: Erythropoietin has different impacts on the cells in normoxia and hypoxia. Its positive influence on fibroblast migration during hypoxia seems to support the strategies of applying erythropoietin in those chronic wounds, which exhibit fibroblastic dysfunction although good vascularisation is present (AU)

Erythropoietin ameliorates the reduced migration of human fibroblasts during in vitro hypoxia.

Erythropoietin promotes the formation of granulation tissue when administered to soft tissue wounds and it was shown to be most effective under tissue hypoxia. However, the action of erythropoietin on the cellular level is not well understood. In order to get a better insight into these processes, an in vitro wound healing assay was applied. Two main players of soft tissue healing-fibroblasts and microvascular endothelial cells-were used as mono- and co-cultures, subsequently inflicting in vitro wounds. Cell migration, proliferation, the differentiation of fibroblasts to myofibroblasts, and the release of vascular endothelial cell growth factor A and angiogenin were quantified in response to hypoxia and erythropoietin (5 IU/ml). Erythropoietin supplementation did neither affect proliferation nor migration of endothelial cells and fibroblasts under normoxia. Under hypoxia, the reduced fibroblast migration was ameliorated by erythropoietin. This effect coincided with an attenuated release of vascular endothelial growth factor A, whereas angiogenin release was unaffected by erythropoietin. The in vitro model applied in this study may represent an adequate approximation to certain aspects of the in vivo status of soft tissue regeneration and the results might serve to interpret the in vivo efficiency of erythropoietin at the cellular level: Erythropoietin has different impacts on the cells in normoxia and hypoxia. Its positive influence on fibroblast migration during hypoxia seems to support the strategies of applying erythropoietin in those chronic wounds, which exhibit fibroblastic dysfunction although good vascularisation is present.

Reoxygenation attenuates the adhesion of neutrophils to microvascular endothelial cells.

In humans, the pathophysiological inflammation response subsequent to hypoxia and reoxygenation often leads to systemic inflammation and multiorgan failure. We applied a newly developed static interaction model using human polymorphonuclear neutrophils and microvascular endothelial cells to clarify the role of hypoxia and hypoxia/reoxygenation in vitro. Human dermal microvascular endothelial cell cultures (n = 7) were exposed to hypoxia and different reoxygenation periods and the adherence rate of neutrophils to the endothelial cells as well as to the protein matrix on the culture slide surface were determined by quantitative microscopy. Hypoxia clearly triggered neutrophil adhesion to human dermal microvascular endothelial cells whereas additional reoxygenation significantly decreased neutrophil adhesion. These in vitro findings suggest that systemic inflammation caused by increased neutrophil adherence to the microvascular endothelium is already initiated by hypoxia rather than by subsequent reoxygenation.

In vitro wounding: effects of hypoxia and transforming growth factor beta1 on proliferation, migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model.

The adequate reconstitution of human soft tissue wounds requires the coordinated interaction of endothelial cells and fibroblasts during the proliferation phase of healing. Endothelial cells assure neoangiogenesis, fibroblasts fill the defect and provide extracellular matrix proteins, and myofibroblasts are believed to support the reconstitution of microvessels. In the present study, we combined in vitro-wound size measurement and multicolour immunocytochemical staining of co-cultured human dermal microvascular endothelial cells and normal human dermal fibroblasts, recently introduced as co-culture scratch-wound migration assay. Applying antibodies for alpha-smooth-muscle actin, von Willebrand factor, extra domain A fibronectin and endothelin-1, we were able to monitor proliferation, migration and the differentiation process from fibroblasts to myofibroblasts as a response to hypoxia. Furthermore, we verified, whether transforming growth factor beta1 (TGFbeta1) and endothelin-1 are able to mediate this response. We show, that proliferation and migration of endothelial cells and fibroblasts decreased under hypoxia. The additional administration of TGFbeta1 did not significantly attenuate this decrease. Solely the myofibroblast population in co-culture adapted well to hypoxia, when cultures were supplemented with TGFbeta1. Considerating the data concerning TGFbeta1 and endothelin-1, we propose a model explaining the cellular interaction during early and late proliferation phase of human wound healing.

Induction of myofibroblastic differentiation in vitro by covalently immobilized transforming growth factor-beta(1).

Growth factors are an important tool in tissue engineering. Bone morphogenetic protein-2 and transforming growth factor-beta(1) (TGF-beta(1)) are used to provide bioactivity to surgical implants and tissue substitute materials. Mostly growth factors are used in soluble or adsorbed form. However, simple adsorption of proteins to surfaces is always accompanied by reduced stability and undefined pharmacokinetics. This study aims to prove that TGF-beta(1) can be covalently immobilized to functionalized surfaces, maintaining its ability to induce myofibroblastic differentiation of normal human dermal fibroblasts. In vivo, fibroblasts differentiate to myofibroblasts (MFs) during soft tissue healing by the action of TGF-beta(1). As surfaces for our experiments, we used slides bearing aldehyde, epoxy, or amino groups. For our in vitro cell culture experiments, we used the expression of alpha-smooth muscle actin as a marker for MFs after immunochemical staining. Using the aldehyde and the epoxy slides, we were able to demonstrate the activity of immobilized TGF-beta(1) through a significant increase in MF differentiation rate. A simple immunological test was established to detect TGF-beta(1) on the surfaces. This technology enables the creation of molecular "landscapes" consisting of several factors arranged in a distinct spatial pattern and immobilized on appropriate surfaces.

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts.

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.

Different cell cycle responses of wound healing protagonists to transient in vitro hypoxia.

Polyploidization is a process present in cells of many different human tissues. Since it is also prominent in human wound healing in vivo and in vitro, we focused on the influence of hypoxia on the cells' proliferation and polyploidization response. The proliferation response of two major cell types, involved in human wound healing, human dermal microvascular endothelial cells (HDMEC) and normal human dermal fibroblasts (NHDF) was quite similar in the in vitro setup: proliferation significantly decreased under the influence of 18 h of hypoxia and was reinitiated after 72 h of reoxygenation. The cells' response concerning their tendency towards the development of polyploidy was different: NHDF did not generate any polyploid cells, which stands in contrast to former in vitro studies with human wound-derived fibroblasts, but HDMEC were characterized by the presence of both mononuclear and binuclear tetraploid cells. The number of tetraploids was downregulated during hypoxia and increased during reoxygenation, accompanied by proliferation onset. The immunomicroscopic survey of HDMEC opened up a cell cycle model, which might be useful in the future to evaluate cell cycle modulations leading to polyploidy without the need to apply any additional cell cycle inhibitors.