Johan P.E. Junker

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.

Tissue Engineering of Skin

Tissue engineering (TE) and regenerative medicine area blend of developmental biology, life sciences, and engi-neering efforts that attempts to address clinical problems.Tissue engineering was defined in 1988 as the applicationof principles and methods of engineering and lifesciences toward fundamental understanding of structurefunction relationships in normal and pathologicalmammalian tissues and the development of biologicsubstitutes to restore, maintain, or improve tissue func-tion

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.

Harnessing growth factors to influence wound healing.

Cutaneous wound healing is a dynamic process with the ultimate goal of restoring skin integrity. On injury to the skin, inflammatory cells, endothelial cells, fibroblasts, and keratinocytes undergo changes in gene expression and phenotype, leading to cell proliferation, migration, and differentiation. Cytokines and growth factors play an essential role in initiating and directing the phases of wound healing. These signaling peptides are produced by a variety of cells and lead to a concerted effort to restore the skin barrier function.

Epidermal regeneration by micrograft transplantation with immediate 100-fold expansion.

Background: Major loss of skin following burns or trauma requires skin grafting for repair. In addition, chronic wounds frequently require skin grafts. Current treatments are either cumbersome, limited in possible expansion ratio, costly, or require extensive time for treatment. This study investigates a new way of regenerating skin after major burns and other trauma, providing 100-fold expansion of a split-thickness skin graft. Methods: Submillimeter micrografts were created by controlled mincing of a split-thickness skin graft and transplanted to porcine full-thickness wounds. By creating an incubator-like microenvironment using wound chambers, the micrografts provide reepithelialization whether placed dermal side up or dermal side down. Results: Transplantation of micrografts in a 1:100 expansion ratio results in complete epithelialization of both healthy and diabetic wounds within 14 days. In comparison, nontransplanted wounds showed 62 percent reepithelialization in healthy pigs and 49 percent in diabetic pigs at the corresponding time point. Conclusions: Minced skin micrografts are very effective in wound repair and can provide 100-fold expansion of a skin graft. Early clinical results confirm the utility of this technique.

Assessing quality of healing in skin: review of available methods and devices.

The process of wound healing is dynamic and takes place over months to years, during which there is a resolution of angiogenesis, continued wound contraction, and connective tissue remodeling. The outcome of this process is most commonly the formation of a scar, defined as a fibrous tissue replacing normal tissues destroyed by injury or disease. Scars often have a lowered or total loss of vital skin functions and imbue a large burden on both the patient and the health care system as a whole. Scar treatments are plentiful but are often unsatisfactory or inconsistent. No single treatment method has been universally adopted. To evaluate the clinical treatment as well as research focused on developing novel methods for scar management, objective studies of the progression of scar formation and the properties of mature scars are needed. Several parameters, including barrier function as well as mechanical and physiological properties, need to be taken into account when both categorizing and treating healing wounds and scars. To date, there is no available methodology that provides a comprehensive evaluation of a scars properties. This review aims at presenting an overview of available scar assessment methods and devices, ranging from analysis of collagen properties in tissue biopsies to noninvasive methods for studies of mechanical parameters such as breaking strength and skin elasticity. In the cases where conclusive studies have been performed, the differences between normal skin and scar with respect to the above parameters are presented. Furthermore, this review highlights areas where the development of additional modalities are needed.

The microenvironment of wound healing.

Abstract This review summarizes experiments performed by us and others, examining the importance of the microenvironment to wound healing. The development of a sealed polyurethane wound chamber has allowed us to perform studies evaluating the effects of growth factors, transplanted cells, and other bioactive substances on wound healing. Studies have compared wet, moist, and dry healing, with the conclusion that a wet, incubator-like microenvironment provides the fastest healing with fewest aberrations and least scar formation. The wet environment is also paramount for the survival and proliferation of transplanted cells or tissue, which has been shown in studies of porcine and human wounds. Moreover, high concentrations of antibiotics and other agents can be introduced in the wound chamber, thereby effectively fighting infection, while maintaining safe systemic concentrations. These findings have been used in clinical settings to treat wounds of different types. A titanium chamber has been developed to create an in vivo incubator, which will serve as a regenerative platform for in vivo tissue engineering.

Moist dressing coverage supports proliferation and migration of transplanted skin micrografts in full-thickness porcine wounds

Transplantation of skin micrografts in a 1:100 ratio regenerate the epidermis of full-thickness wounds in pigs within 14 days in a wet environment. The aim of the current study was to combine micrografts and commercially available moist dressings. We hypothesized that micrografts regenerate the epidermis when covered with a moist dressing. 5cm×5cm and 10cm×10cm full-thickness wounds were created on the backs of pigs. Wounds were transplanted with 0.8mm×0.8mm micrografts created from a split-thickness skin graft in a 1:100 ratio. 5cm×5cm wounds were treated with wound chambers, moist dressings or dry gauze (non-transplanted control group). 10cm×10cm wounds were compared to non-transplanted wounds, both covered with moist dressings. Reepithelialization was assessed in biopsies from day 10, 14 and 18 post-transplantation. 5cm×5cm transplanted wounds covered with moist dressings showed 69.5±20.6% reepithelialization by day 14 and 90.5±10.4% by day 18, similar to wounds covered with a wound chamber (63.9±16.7 and 86.2±11.9%, respectively). 18 days post-transplantation, 10cm×10cm transplanted wounds covered with moist dressings showed 66.1±10.3% reepithelialization, whereas nontransplanted wounds covered with moist dressings were 40.6±6.6% reepithelialized. We conclude that micrografts combined with clinically available moist dressings regenerate the epidermis of full-thickness wounds.

Transplantation of acellular dermis and keratinocytes cultured on porous biodegradable microcarriers into full-thickness skin injuries on athymic rats

In search of an optimal transplantation regime for sufficient dermal and epidermal regeneration after a full-thickness skin injury, wounds on athymic rats were grafted with split-thickness skin grafts or acellular human dermis followed by transplantation with human keratinocytes either in single-cell suspension or cultured on porous biodegradable microcarriers. After 2 weeks, all wounds grafted with acellular human dermis showed a well organised and vascularised dermal component and reepithelialisation on the grafted dermal matrix was complete 21 days after transplantation with human keratinocytes. Wounds grafted with human keratinocytes seeded on biodegradable microcarriers or split-thickness skin grafts displayed over time (i.e. 16-21 days post-transplantation) a significantly thicker epithelial cell layer in comparison to wounds grafted with keratinocytes in single-cell suspensions or microcarriers not seeded with cells. Furthermore, measurements of dermal thickness in the closed wounds 21 days after grafting showed a significantly thicker and well organised neodermal component in wounds transplanted with keratinocytes seeded on microcarriers or split-thickness skin grafts compared to all other wounds. Positive immunostaining towards von Willebrand factor revealed the plausible proangiogenic effects of transplantation with keratinocytes seeded on microcarriers. Analysis of representative tissue sections after fluorescence in situ hybridisation visualised that grafted human keratinocytes were present in the epidermal layers covering the wounds 16 and 21 days after transplantation, strongly indicating preservation of cell viability. These results shows that the use of biodegradable microcarriers in the culture of autologous keratinocytes for treatment of full-thickness wounds not only facilitate the cultivation, transportation and transplantation processes but also enhances the dermal regeneration induced by a dermal scaffold which results in a clinical result that is significantly superior to the one obtained when keratinocytes are transplanted in a single-cell suspension.