A Scientific essay in Medical Sciences DOCTORAL THESIS To obtain the degree of doctor from the Radboud University of Nijmegen on the authority of Rector Magnificus, Prof. dr. C.W.P.M Blom, according to the decision of the Council of Deans to be defended in public on Monday 16th January, 2006 at 3.30 a.m. By: Hugo Egidius van Beurden Born in Drunen on April 9th, 1971

Chapter 1 [contents]
 

Disturbances in maxillary development and dento-alveolar growth after cleft palate repair have been a problem for surgeons for a long time (Perko, 1986). In the first half of the 19^th century many surgeons attempted to optimize cleft palatal closure(Zigiotti et al., 1983), but in spite of all the efforts, serious growth problems remained(Millard, 1976). Since that time, some progress has been made by modifying surgical techniques. Only in the eighties of the last century it was shown that wound contraction and scar tissue formation formed the basis of the inhibition of maxillary development and dento-alveolar growth(Kremenak, 1984).

Fibroblasts and myofibroblasts are important cells in wound healing. They mediate wound contraction and scar formation, and therefore, interference with fibroblast activity might decrease the growth disturbances. To achieve this, the wound fibroblasts first have to be characterized. This was the main subject of this thesis. More precisely, the aims of this study were:

(1)    to characterize fibroblasts at different stages of wound healing in vivo and also after  in vitro culture. Accumulating evidence shows that fibroblasts are dynamic cells occurring in functionally and morphologically heterogeneous subpopulations. It was proposed that also during oral wound healing distinct fibroblast subpopulations occur (Lekic et al., 1997). We investigated whether such subpopulations are also present during palatal wound healing. Therefore, we analyzed the expression of integrins, cytoskeletal proteins, and the cytoprotective enzyme heme oxygenase-1 in vitro and in vivo. Additionally, we investigated changes in cell density and apoptosis in vivo.

(2)    to relate the fibroblast phenotypes to functional activity in vitro. Fibroblasts migrate into the wound and proliferate in response to fibronectin and several growth factors secreted by neutrophils and macrophages(Singer and Clark, 1999). Adhesion and migration of these cells are essential for the formation of granulation tissue(Singer and Clark, 1999). Furthermore, wound contraction is initiated by migrating fibroblasts generating mechanical tension(Darby et al., 1990). Therefore, we compared the adhesion and migration of fibroblasts from early and late phases of wound healing with age-matched cells from unwounded palates.

Chapter 2  [contents]

In chapter 2, we discussed several theoretical options for the modulation of myofibroblasts. More feasible options focus on the reduction of myofibroblasts numbers, either by inhibition of their differentiation, or by stimulation of their apoptosis. The latter might be achieved by the local application of certain growth factors but more information of the exact signals inducing myofibroblasts apoptosis is required. Inhibition of myofibroblast differentiation me be achieved by the blocking of ED-A fibronectin, one of the essential factors for myofibroblast differentiation. This protein is specific for the wound healing process, and as such is not expected to induce side effects elsewhere in the body.   

Chapter 3  [contents]

Experimental in vivo models

Since the breeding of animals with standardized clefts appears to be impossible, animals with surgically created clefts have been used. Cats, dogs, rats, rabbits, and primates have been used to study the different aspects of a cleft palate (Bardach and Kelly, 1988).  To study the effects of surgery on growth, mainly dogs, cats or primates were employed. These animal models, however, are very expensive, and species-specific antibodies are often not available. This makes them less suitable for cell biological research. Rats provide a more convenient model, since they are relatively cheap and many antibodies against rat integrins and cytoskeletal markers are available. Drawbacks of the rat as a model are the differences existing between wound healing in rats and humans and that rats are too small to perform proper palatal surgery. The latter means that the effect of surgery on long term growth cannot be studied in the rat model. Therefore, these aspects have to be studied in animals that mimic the human situation more closely.

Since the main aim of this thesis is the characterization of the fibroblasts during palatal wound healing, a rat model is suitable. Palatal full-thickness wounds made with a biopsy punch are thought to mimic the wounds resulting from cleft palate surgery in humans. The feasibility of this method has already been demonstrated (Cornelissen et al., 1999).

Although the palatal wound healing process is scarcely studied, it appears to be comparable to the extensively described dermal wound healing process. Rat dermal wound healing does not perfectly mimic human dermal wound healing since the skin morphology is different (Marx and Mou, 2002). Rats are described as loose-skinned animals, while humans have a "tight" skin. This "loose" skin allows wound contraction to play a more important role in dermal wound closure, causing a shorter overall healing time in rats than in humans. This difference complicates the comparison between the two species(Davidson, 1998). However, the more fundamental aspects of wound healing are largely comparable. In summary, the rat model is suitable to study the more fundamental biological aspects of palatal wound healing.

Experimental in vitro models

The same rat model as described above provides material for the in vitro studies. Fibroblasts are cultured from full-thickness wound tissue, obtained with a biopsy punch at different time points during wound healing. These cell cultures are used to study the specific characteristics of the fibroblasts at different stages. An advantage of cell culture studies is that large numbers of cells are available after propagation. However, a prerequisite is that the phenotype of these cells is stable during culture. Since we observed marked differences in fibroblast phenotypes depending on the collection time after wounding we conclude that the phenotype of the different fibroblast populations remains stable in culture (see chapter 3). Therefore, we assume that the cultured cells indeed represent the fibroblastic phenotypes characteristic for different stages of the wound healing process. The expression profile of fibroblasts in situ was similar to that found after in vitro culturing adding evidence that indeed the different fibroblasts populations are stable in culture.

Study design

To characterize the fibroblast subpopulations, tissue samples from different phases of the wound healing process were used. The sampling protocol is schematized in figure 1. At t=0, biopsies from the unwounded tissue were obtained, after which the wounds were allowed to heal. At 3, 5, 8, 15, 30, 60, or 90 days after wounding biopsies were taken from the healing tissue and either processed for western blotting and immunohistochemistry, or for fibroblast culture. For the in vitro experiments, biopsies were cut into pieces and the fibroblasts were cultured for four passages. These cells were used for flow cytometry (FACS), western blotting of whole cell lysates, immunocytochemical analysis, and for adhesion and migration assays.

Figure 1: Sample protocol.

Analytical parameters

The analytical markers used in this study are summarized in table 1.

Integrins

The a₁b_1, a₂b₁ and a₅b₁ integrins are important for the transmission of forces over the wound and for interactions between fibroblasts and collagen during the remodeling phase(Welch et al., 1990; Schiro et al., 1991). The a_v subunit can combine with several b subunits to serve as receptors for vitronectin and fibronectin(Gailit and Clark, 1994). Finally, the a₆ subunit is included because it is thought to be unique for anchoring basal keratinocytes to the basement membrane(Sonnenberg et al., 1991), but whether it is also expressed by fibroblasts is still a matter of debate.

Cytoskeletal markers

a-Smooth muscle actin (a-SMA) belongs to the actin microfilament system and enables myofibroblasts to generate forces during the contraction of granulation tissue(Gabbiani, 1992). The intermediate filament vimentin also contributes to the generation of these forces(Coleman and Lazarides, 1992). Vinculin, is an important component of focal adhesion complexes(Bailly, 2003).

Enzyme

Heme oxygenase-1 is an important cytoprotective enzyme during wound healing because it neutralizes the toxic heme that is released from heme proteins after wounding  (Willis, 1995; Brouard et al., 2000; Duckers et al., 2001).

Cell density

Apoptosis and DNA content are used as measures for changes in cellularity.

Functional assays

Migration and adhesion are determined, since both are essential for wound contraction and scar tissue formation (Nedelec et al., 2000).

Table 1:  Markers included in the study.

Chapter 3 and 4  [contents]
 

Results

Analysis of fibroblast parameters in vitro and in vivo

The expression of integrin subunits was analyzed during palatal wound healing in vivo, and in cultured cells from the different stages (chapters 3 and 4). All subunits showed remarkable changes in expression both in vitro and in vivo during the first two weeks of palatal wound healing. This dynamic period may be related to inflammation, re-epithelialization, wound contraction, and the formation of a provisional matrix, since all these processes require rapid changes in integrin expression. The integrin subunits a₁ and b₁ also showed an extended increase in expression later in wound healing. Although at that time "less active" fibroblasts were observed with a more stable phenotype, the unwounded situation was never re-established. This indicates that true healing is not achieved during the time frame of the present studies, and that scar tissue may remain in the wound.

The expression of some important cytoskeletal markers was also analyzed in the fibroblast populations in vitro, and in vivo (chapters 3 and 4). Shortly after wounding, the amount of vimentin increased in tissue homogenates. However, the number of vimentin-positive fibroblasts did not change during wound healing. This indicates that the amount of vimentin per cell is increased rather than the number of vimentin-positive cells. The in vivo expression of a-SMA by fibroblasts also showed a maximum around eight days post wounding, after which it decreased to below the level of the unwounded control. The in vitro expression of a-SMA in the isolated fibroblast populations corresponded highly with these data. Thus, most myofibroblasts are present around eight days post- wounding. Thereafter, the myofibroblasts disappear. The early peak in myofibroblast numbers is also described by others(Desmouliere and Gabbiani, 1996). The expression of vinculin, which was used as a marker for focal adhesions, was high in unwounded tissue, significantly lower early in wound healing, and again high later in wound healing. The number of focal adhesions appears to be inversely related to the motility of cells(Huttenlocher et al., 1995; Murphy-Ullrich, 2001). Our data therefore can be interpreted as a low fibroblast motility in unwounded tissue, a high motility early in wound healing, and a reduced motility later on.

Chapter 5  [contents]
 

The results from the functional assays confirm the above findings. Adhesion and migration of wound fibroblasts from fibroblast populations isolated in the course of palatal wound healing were studied (chapter 5). Sessile fibroblasts obtained from unwounded control tissue showed low migration activity and high adhesion in vitro. In contrast, fibroblasts obtained later in wound healing showed a much higher migration and less adhesion. Adhesion and migration are indeed shown to be inversely related (Couchman and Rees, 1979; Dunlevy and Couchman, 1993).

Apart from the changing expression patterns of integrins and cytoskeletal proteins, we also observed changes in cell density during palatal wound healing, however none of these changes were significant (chapter 4). The maximum cell density was observed between one and two weeks after injury which corresponded with the time of maximum granulation tissue formation and wound contraction. We also observed a maximum in the number of apoptotic cells around two weeks after injury. The decrease in cellularity during wound healing is generally believed to be caused by apoptosis (Desmouliere, 1995). Indeed, we observed a maximum in the number of apoptotic cells around two weeks after injury, after which the number of apoptotic cells decreased (chapter 4).

Chapter 6  [contents]
 

Heme oxygenase expression of fibroblasts in vitro and in vivo

During palatal wound healing, fibroblasts showed specific changes in in vivo and in vitro expression patterns of the heme degrading enzyme heme oxygenase-1 (HO-1) (chapter 6). Early in wound healing the number of HO-1 positive fibroblasts and the average expression per cell was strongly increased. Late in wound healing, these levels returned to the normal unwounded levels. HO-1 has a cytoprotective role early in wound healing during the inflammatory phase (Kampfer et al., 2001). Later in wound healing, when the inflammation subsides, HO-1 expression returns to normal. Recent data also indicate antifibrotic activities of HO-1 later in wound healing(Li et al., 2003).

Chapter 7  [contents]
 

Conclusions

From these studies we can conclude that heterogeneous populations of fibroblasts are present in the course of  palatal wound healing. These different subpopulations can be isolated and cultured, and maintain their characteristics up to seven passages. We distinguished two main fibroblast phenotypes both during palatal wound healing in vivo, and in vitro: an early "activated" phenotype and a late more "quiescent" phenotype. These fibroblast phenotypes were also observed in the functional assays for adhesion and migration.

The modulation of activity of specific fibroblast phenotypes might offer a good starting point for the control of wound contraction and scar formation.

Future Research

The discovery of specific fibroblast populations during wound healing offers opportunities for future manipulation of the wound healing process. As these populations were shown to be maintained in culture, further characterization can be carried out in functional wound healing models. A first attempt was made in chapter 5 with the study of adhesion and migration of fibroblast populations. Further studies, for example in three-dimensional contraction models, should focus on myofibroblasts, the main cells responsible for contraction and scarring. In addition, functional models can be used to develop methods for the modulation of (myo)fibroblast activity.

The emphasis of this study was on the role of fibroblast integrins in palatal wound healing. Although we characterized the expression of several integrins on palatal fibroblasts, it will be difficult to modulate palatal wound healing by intervention on the integrin level. The same integrins present on other fibroblasts or even on other cell types will also be affected.