Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells: Are mesenchymal stromal cells involved in scar formation?

In this work, different fibroblast-like (mesenchymal) cell populations that might be involved in wound healing were characterized and their involvement in scar formation was studied by determining collagen synthesis and processing. Depending on the physical and mechanical properties of the tissues, specific collagen cross-linking routes are followed. In skin the cross-linking of the pyridinium type is normally very low; however, in different forms of fibrosis increased levels of this type of cross-linking have been found. The enzyme lysyl hydroxylase-2b (LH-2b) plays a crucial role in this type of cross-linking. The gene expression levels of LH-2b, alpha-smooth muscle actin, and collagen types I and III were determined in dermis, subcutaneous fat, and (hypertrophic) scar tissue as well as in isolated cultured mesenchymal cells derived from these tissues, by real-time RT-polymerase chain reaction. Cultured mesenchymal cells from fat and scar tissue as well as the tissues itself showed significantly higher expression of LH-2b, alpha-SMA, and collagen type I than dermal mesenchymal cells. LH-2b-dependent pyridinium cross-linking was significantly enhanced in fat and scar tissue compared with dermis. FACS analysis was performed to characterize the fibroblast-like cells from the dermis, fat, and scar tissue. All cell populations express the distinct pattern of CD markers also expressed by mesenchymal stromal cells. Furthermore, parts of these cell populations were able to differentiate into adipocytes, chondrocytes, and osteoblasts. We conclude, therefore, that mesenchymal (stem) cells from the subcutaneous fat might be responsible for the accumulation of collagen in these scars.

Culture of keratinocytes for transplantation without the need of feeder layer cells.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Expression profile of proteins involved in scar formation in the healing process of full-thickness excisional wounds in the porcine model.

Scar formation in deep dermal wounds is associated with excessive collagen deposition and contraction. Increased collagen synthesis and decreased collagen degradation are the mechanisms through which this form of fibrosis can occur. Another factor might be a different kind of collagen cross-linking seen in fibrotic skin diseases. This type of cross-linking is dependent on the enzyme lysyl hydroxylase-2b. In this study, we examined the expression profile of the potential key players in scar formation in time in healing of acute wounds. Collagen types I and III, lysyl hydroxylase-2b, alpha-smooth muscle actin, transforming growth factor betas, and the matrix metalloproteinases and their inhibitor mRNA levels were determined. All genes examined show distinct expression patterns over time. The expression of lysyl hydroxylase-2b peaks at day 7, and precedes collagen types I and III expression. Eight weeks after wounding, the scars showed an increased level of lysyl hydroxylase-2b-mediated collagen cross-linking. This study shows that the fibrosis-specific type of cross-linking of collagen seen in human hypertrophic scarring also plays a role in this animal model of wound healing. Moreover, the expression of the putative gene responsible for this type of cross-linking, the lysyl hydroxylase-2b, is elevated during wound healing.

Culture of Keratinocytes for Transplantation without the Need of Feeder Layer Cells.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Differential expression of CRABP-II in fibroblasts derived from dermis and subcutaneous fat.

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.

Upside-down transfer of porcine keratinocytes from a porous, synthetic dressing to experimental full-thickness wounds.

Currently, the use of cultured epithelial autografts as an alternative to split-thickness skin autografts for coverage of full-thickness wounds is limited due to fragility of the sheet and variability in the outcome of healing. This could be circumvented by the transfer of proliferating keratinocytes, instead of differentiated sheets, to the wound bed and the "in vivo" regeneration of epidermis. The aim of this study was to achieve re-epithelialization on experimental full-thickness wounds in the pig using a porous, synthetic carrier seeded with proliferating keratinocytes. Porcine keratinocytes were isolated by enzymatic digestion and cultured in Optimem basal medium with mitogens. In a full-thickness wound model, carriers with different seeding densities were transplanted upside down onto the wound bed. Keratinocytes were labeled using a fluorescent red membrane marker, PKH-26 GL. Transfer of keratinocytes and re-epithelialization were recorded macroscopically and histologically. On day 4 after transplantation, transfer of fluorescently labeled keratinocytes was shown by their presence in the granulation tissue. An immature epidermis, as well as epithelial cords and islands, formed as early as day 8. At day 12 a stratified epidermis and wound closure were established and epithelial cysts were formed by differentiation of epithelial islands. Wounds treated with seeding densities as low as 50,000 cells/cm(2) showed wound closure within 12 days, whereas wounds treated with 10,000 cells/cm(2) or the nonseeded (acellular) carriers did not show complete re-epithelialization before day 17 after treatment. This study showed that porcine keratinocytes, transplanted "upside down" in experimental full-thickness wounds using a synthetic carrier, continued to proliferate and started to differentiate, enabling the formation of a new epidermis in a time frame of 12 days.