Gunnar Kratz

Heparin-chitosan complexes stimulate wound healing in human skin.

The effect of heparin ionically linked to chitosan on the stimulation of re-epithelialisation of full thickness wounds in human skin was investigated in an in vitro model. After seven days of incubation, heparin-chitosan gel stimulated 9/10 of the full thickness wounds to re-epithelialise compared with only 3/10 of the wounds that were covered with chitosan gel or membrane, and none of the wounds incubated without gel or membrane or with heparin solution alone. Both dermal and epidermal cells were viable after the incubation time. Furthermore, the stimulatory effect of the heparin-chitosan complexes depended on the concentration of heparin in the complex. We hypothesise that these effects are caused by stabilisation and activation of growth factors that bind to immobilised heparin.

Fibroblasts derived from human chronic diabetic wounds have a decreased proliferation rate, which is recovered by the addition of heparin

We have studied the growth kinetics of fibroblasts derived from uninjured skin and chronic wounds in non-diabetic and diabetic (IDDM) patients. DNA measurements during the first 24 h after cell starvation showed that fibroblasts derived from chronic wounds, both non-diabetic and diabetic, display a decreased adhesion and proliferation. When determining the rate of proliferation after another 48, 72 and 96 h, a significant decrease in the proliferation rate was found in the chronic wound fibroblasts compared to those from uninjured skin. Furthermore, we have investigated the effects of heparin, hyaluronic acid and other heparin-like substances on the proliferation of non-diabetic and diabetic fibroblasts. We found that these substances stimulated the proliferation of human fibroblasts derived from both normal skin and chronic wounds measured as DNA content. Stimulation with heparin normalized the proliferation of the diabetic chronic wound fibroblasts. This effect was independent of the presence of serum. The effect of heparin was dose-dependent and most pronounced during the first 24 h of stimulation. These results suggest that heparin may be of importance in the treatment of chronic diabetic wounds.

Salinomycin induces activation of autophagy, mitophagy and affects mitochondrial polarity: Differences between primary and cancer cells☆

The molecular mechanism of Salinomycins toxicity is not fully understood. Various studies reported that Ca(2+), cytochrome c, and caspase activation play a role in Salinomycin-induced cytotoxicity. Furthermore, Salinomycin may target Wnt/β-catenin signaling pathway to promote differentiation and thus elimination of cancer stem cells. In this study, we show a massive autophagic response to Salinomycin (substantially stronger than to commonly used autophagic inducer Rapamycin) in prostrate-, breast cancer cells, and to lesser degree in human normal dermal fibroblasts. Interestingly, autophagy induced by Salinomycin is a cell protective mechanism in all tested cancer cell lines. Furthermore, Salinomycin induces mitophagy, mitoptosis and increased mitochondrial membrane potential (∆Ψ) in a subpopulation of cells. Salinomycin strongly, and in time-dependent manner decreases cellular ATP level. Contrastingly, human normal dermal fibroblasts treated with Salinomycin show some initial decrease in mitochondrial mass, however they are largely resistant to Salinomycin-triggered ATP-depletion. Our data provide new insight into the molecular mechanism of preferential toxicity of Salinomycin towards cancer cells, and suggest possible clinical application of Salinomycin in combination with autophagy inhibitors (i.e. clinically-used Chloroquine). Furthermore, we discuss preferential Salinomycins toxicity in the context of Warburg effect.

Dose-dependent hyperbaric oxygen stimulation of human fibroblast proliferation

Diabetic wounds are characterized by a prolonged wound healing process with insufficient formation of granulation tissue. Systemic hyperbaric oxygen therapy has been observed to improve the healing of these wounds. However, the mechanism(s) responsible for these findings are not yet fully elucidated. In the present study we have studied the in vitro effects of hyperbaric oxygen on proliferation of human fibroblasts from normal skin and from chronic foot ulcers in non‐insulin‐dependent diabetics. A 1‐hour exposure to hyperbaric oxygen at oxygen pressures between 106 and 300 kPa (795 to 2250 mm Hg) increased the proliferation in both diabetic and normal fibroblasts. The stimulatory effect was dose‐dependent, with a peak increase in cell proliferation at 250 kPa and 200 kPa for normal and diabetic cells, respectively. The effects were not due to hydrostatic pressure per se. These results suggest that hyperbaric oxygen could stimulate fibroblast activity in the diabetic wound, a finding that could explain the enhanced formation of granulation tissue seen clinically in wounds treated with hyperbaric oxygen. We also speculate that mechanisms other than just increased oxygen availability may be responsible for our findings.

Adipogenic, Chondrogenic and Osteogenic Differentiation of Clonally Derived Human Dermal Fibroblasts

The apparent need of an autologous cell source for tissue engineering applications has led researchers to explore the presence of cells with stem cell plasticity in several human tissues. Dermal fibroblasts (FBs) are easy to harvest, expand in vitro and store, rendering them plausible candidates for cell-based therapies. The aim of the present study was to observe the effects of adipogenic, chondrogenic and osteogenic induction media on the phenotype of human FBs. Human preadipocytes obtained from fat tissue have been proposed as an adult stem cell source with suitable characteristics, and were used as control cells in regard to their differentiation potential. Routine staining, immunohistochemical analysis and alkaline phosphatase assay were employed, in order to study the phenotypic shift. FBs were shown to possess multilineage potential, giving rise to fat-, cartilage- and bone-like cells. To exclude contaminant progenitor cells or cell fusion giving rise to tissue with adipocyte-, chondrocyte- and osteoblast-like cells, single-cell cloning was performed. Single-cell-cloned FBs (sccFBs) displayed a similar differentiation potential as primary-culture FBs. The presence of ‘stem-cell-specific’ surface antigens was analyzed using flow cytometry. The results reveal that sccFBs have several of the markers associated with cells exhibiting stem cell plasticity. The findings presented here are corroborated by the findings of other groups, and suggest the use of human dermal FBs in cell-based therapies for the reconstruction of fat, cartilage and bone.

Mechanical tension stimulates the transdifferentiation of fibroblasts into myofibroblasts in human burn scars

Scar formation as a result of burn wounds leads to contraction of the formed granulation tissue, which causes both aesthetic and functional impairment for the patient. Currently, the main treatment methods focus on stretching to prevent tissue contraction. The myofibroblasts play a key role in the contraction of granulation tissue during scar formation, but their presence should normally decrease after wound re-epithelialization. In hypertrophic scars the myofibroblasts persist and is believed to cause further hypertrophy. Previous studies have shown that mechanical tension leads to increased myofibroblast numbers in granulation tissue. In order to evaluate the effect mechanical tension as a result of stretching has on the number of myofibroblasts in burn wound scars, an in vitro model was used. This model used human burn scar biopsies which were stretched and examined after 1 and 6 days to evaluate the effect on the number of myofibroblasts. The stretching caused an increase in the number of myofibroblasts after mechanical stimulation. This indicates that mechanical stimulation using stretching induces fibroblast to myofibroblast transdifferentiation, thus underlining the importance of further investigations of optimal methods of this regime for treating burn scars.

Mammary Epithelial Cell and Adipocyte Co-Culture in a 3-D Matrix: The First Step towards Tissue-Engineered Human Breast Tissue

Reconstruction of the female breast after cancer surgery is a demanding task where the methods used today suffer from several disadvantages. In the present study we have investigated the possibility to use tissue engineering methods to regenerate human autologous breast tissue. Human mammary epithelial cells and preadipocytes were derived from breast tissue biopsies from healthy women undergoing reduction mammoplasty, and the two celltypes were co-cultured with conventional cell culture methods as well as in 3-D matrices. The study shows that it is possible to harvest both human mammary epithelial cells and preadipocytes in a single session, propagate several subcultures, and that the cells maintain a normal intercellular distribution and growth-pattern when co-cultured in a 3-D collagen gel. We propose that growth and formation of a tissue closely resembling normal human breast tissue be readily obtained in the described in vitro cell culture set-up using basic tissue engineering principles. This concept may be of great importance in the development of new methods for reconstruction of the human breast.

Insulin like growth factor-1 and -2 and their role in the re-epithelialisation of wounds; interactions with insulin like growth factor binding protein type 1.

Insulin like growth factor (IGF) 1 and 2 which are present and actively synthesised in the wound fluid stimulate several cell types involved in the process of wound healing. To investigate the role of IGF-1 and 2 and in addition, the association between IGF and their carrier proteins, IGF binding proteins (IGFBP), we have used a newly established model for human wound healing in fresh biopsy material. Histological examination shows that IGF-1 stimulates efficient reepithelialisation of the wounds both alone and in the presence of recombinant IGFBP-1. In contrast, IGF-2 stimulates healing only when used in combination with IGFBP-1. These findings suggest that the two IGFs and their carrier proteins may function during different phases of wound healing and that both IGF-1 and 2 act as potent inducers of wound healing; this may have direct clinical implications.

Engineering three-dimensional cartilage- and bone-like tissues using human dermal fibroblasts and macroporous gelatine microcarriers

The creation of tissue-engineered cartilage and bone, using cells from an easily available source seeded on a suitable biomaterial, may have a vast impact on regenerative medicine. While various types of adult stem cells have shown promising results, their use is accompanied by difficulties associated with harvest and culture. The proposed inherent plasticity of dermally derived human fibroblasts may render them useful in tissue-engineering applications. In the present study, human dermal fibroblasts cultured on macroporous gelatine microcarriers encapsulated in platelet-rich plasma into three-dimensional constructs were differentiated towards chondrogenic and osteogenic phenotypes using specific induction media. The effect of flow-induced shear stress on osteogenic differentiation of fibroblasts was also evaluated. The generated tissue constructs were analysed after 4, 8 and 12 weeks using routine and immunohistochemical stainings as well as an enzyme activity assay. The chondrogenic-induced tissue constructs were composed of glycosaminoglycan-rich extracellular matrix, which stained positive for aggrecan. The osteogenic-induced tissue constructs were composed of mineralised extracellular matrix containing osteocalcin and osteonectin, with cells showing an increased alkaline phosphatase activity. Increased osteogenic differentiation was seen when applying flow-induced shear stress to the culture. Un-induced fibroblast controls did not form cartilage- or bone-like tissues. Our findings suggest that primary human dermal fibroblasts can be used to form cartilage- and bone-like tissues in vitro when cultured in specific induction media.

Adipose tissue processed for lipoinjection shows increased cellular survival in vitro when tissue engineering principles are applied.

Correcting soft tissue defects by autologous fat grafting is a routine procedure in plastic surgery. Its efficacy and safety has been discussed extensively and several techniques of lipoinjection have been developed. However, one is bound to overcorrect by 30%-70% or need to repeat the procedure because of resorption of the transplant. The reasons are that many of the transplanted cells are already differentiated, and also that there is no nutritional support to the inner cell layers when they are transplanted as fragments. By culturing autologous adipocytes one can ensure that only non-differentiated, but committed, preadipocytes are transplanted and the procedure can be done in a way that ensures optimal nutritional support for the cells. In the present study we have compared our cell culture technique with two common clinical ways of processing liposuction material and found that (pre)adipocytes survive and proliferate significantly better in cell culture.