Figure 1.
Dermal biopsy locations from healthy controls and keloid patients with corresponding histology.
A. Transverse view of biopsy locations from normal dermal scar tissue and adjacent normal dermal (non-wounded) skin from which in vitro primary cell cultures were subsequently established. B. Transverse view of marginal peri-lesional and reticular dermal intra-lesional biopsy sites from the keloid scar. C. Cross-section of keloid scar indicating depth of peri-lesional and intra-lesion biopsies. D. Representative H&E staining of tissue section from normal skin indicating organised wavy deposition of collagen (arrows). E. Representative H&E staining of tissue section from a normal scar. F. Representative H&E staining of a peri-lesional keloid tissue section indicating a thickened EP with increased cell infiltration (yellow arrow) and deposition of hyalinised collagen bundles in the RD (black arrow). G. Representative H&E staining of an intra-lesional keloid tissue section indicating thick compact hyalinised collagen bundle deposition in the RD (black arrow). EP = Epidermis, PD = Papillary dermis, RD = Reticular dermis. All the H&E micrographs (D–G) were taken at 200 magnifications.
Table 1.
Demographic data for keloid patients and control subjects.
Figure 2.
Schematic timeline for collection of primary culture conditioned media.
Conditioned media was collected every ∼60 hrs when PKF (n = 5), IKF (n = 5), NF (n = 4) and NS (n = 4) were in proliferative log phase of growth. For each individual primary cell culture, conditioned media was collected at ∼50%, 70% and 90% confluency states and subsequently amalgamated prior to treatment of cells. PKF = peri-lesional keloid fibroblasts, IKF = intra-lesional keloid fibroblasts, NF = normal skin fibroblasts, NS = normal dermal scar fibroblasts.
Figure 3.
Schematic timeline for treatment of primary culture cells with conditioned media.
NF (n = 4), NS (n = 4), PKF (n = 5), and IKF (n = 5) were independently treated with all the different conditioned media types (n = 16) following 24 hrs cell synchronisation. Conditioned media was continually replaced every 60 hrs up to a final treatment period of 240 hrs. RTCA on a micro-sensory array was undertaken for the complete 240 hrs whereas end-point assays were conducted at either 120 hrs or 240 hrs. PKF = peri-lesional keloid fibroblasts, IKF = intra-lesional keloid fibroblasts, NF = normal dermal fibroblasts, NS = normal dermal scar fibroblasts, RTCA = Real time cell analysis.
Table 2.
List of 1° Antibodies used in this study.
Table 3.
List of 2° Antibodies used in this study.
Table 4.
Quantitative real time polymerase chain reaction (qRT-PCR) primers.
Figure 4.
Real Time Cell Analysis (RTCA) over 240 hrs.
A. Increased CI was observed at 60–240 hrs. PKF and IKF media treatment both elicited higher CI than NF media between 60–240 hrs. B. Similar trend for NS treated with PKF, IKF and NS control media was observed but with PKF media eliciting maximum CI at 120 hrs. C. Differences in CI were smaller for PKF with all media treatments, although overall CI values were greater at 60 hrs. CI = cell index, NF = normal dermal fibroblasts (n = 4), NS = Normal dermal scar fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5), IKF = intra-lesional keloid fibroblasts (n = 5).
Figure 5.
Proliferation after 120(day-5) and 240 hrs (day-10) of conditioned media treatment.
A. Significantly increased (*p<0.03) proliferation and cellular viability was observed in both NF and NS treated with PKF or IKF media versus respective control media after 120 hrs. B. Similar trends were observed after 240 hrs although overall proliferation levels were higher than corresponding treatments at 120 hrs. C. Significantly higher proliferation was observed in PKF and IKF when treated with PKF or IKF media versus NF or NS media after 120 hrs. D. Similar trends were observed for PKF and IKF cells at 240 hrs with overall proliferation higher than corresponding treatments at 120 hrs. NF = normal dermal fibroblasts (n = 4), NS = Normal dermal scar fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5), IKF = intra-lesional keloid fibroblasts (n = 5). Significantly increased (†p<0.02) proliferation and cellular viability was also observed in both PKF and IKF treated with PKF or IKF media versus respective NF and NS when treated with NF and NS media.
Figure 6.
Cell migration in scratch wound invasion assay after 240 hrs of conditioned media treatment.
A. Significantly increased (*p<0.03) cell migration occurred in NF over a 30 hrs period following 240 hrs treatment with PKF or IKF conditioned media versus NF control media. B. Significantly increased migration into a scratch wound was also observed in NS treated with PKF and IKF versus NS control media. C. PKF treated with NF control media formed a mesh-like network of cells in the scratch wound and significantly (p<0.01) increased migration following treatment with PKF and IKF conditioned media as compared to normal skin fibroblasts condition media. D. Significant (p<0.03) increased migration was also observed in IKF cells following treatments with PKF or IKF media versus NF control media. Blue = nuclei, Green = phalloidin stained intermediate filaments. NF = normal dermal fibroblasts (n = 4), NS = Normal dermal scar fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5), IKF = intra-lesional keloid fibroblasts (n = 5).
Figure 7.
Cellular organisation in post-confluent in vitro primary cultures.
A. Keloid primary fibroblasts derived from the peri-lesional margin of four keloid scar biopsies (PKF) passage ≤3. Arrows indicate whirl-like ridges and nodular aggregates formed when cells were grown to a post-confluent (>100%) state. B. Normal skin primary fibroblasts (NF) grown to a post-confluent (>100%) state did not show ridge structures or aggregates but grew in parallel layers.
Figure 8.
Protein expression after 240 hrs of replenishing conditioned media treatment.
A. Significantly increased (*p<0.04) expression of collagen I, fibronectin, α-SMA, CTGF, PAI-1 and TGFβ-2 was observed in NF treated with PKF or IKF conditioned media versus NF control media after 240 hrs. Representative triplicates are shown below graphs (green) normalised against α-tubulin (red). B. Increased expression was observed for NS treated with PKF or IKF media versus NS control media after 240 hrs. C. PKF treated with PKF or IKF media elicited higher expression of all protein markers in comparison to both NF and NS media at 240 hrs. D. IKF treated with PKF or IKF media also increased expression compared to both NF and NS media at 240 hrs. NF = normal dermal fibroblasts (n = 4), NS = Normal dermal scar fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5), IKF = intra-lesional keloid fibroblasts (n = 5). Significantly increased (†p<0.04) expression of collagen I, fibronectin, α-SMA, CTGF, PAI-1 and TGFβ was also observed in both PKF and IKF treated with PKF or IKF media versus respective NF and NS when treated with NF and NS media.
Figure 9.
Gene expression measured by quantitative real time polymerase chain reaction (qRT-PCR) after 240 hrs of replenishing conditioned media treatment.
A. Significantly increased (*p<0.03) expression of collagen I, fibronectin, α-SMA, CTGF, PAI-1 and TGFβ mRNA was observed in NF treated with PKF or IKF conditioned media (versus NF control conditioned media) after 240 hrs. All mRNA expression was normalised to the internal reference gene RPL-32. B. Increased gene expression was observed for NS treated with PKF or IKF conditioned media versus NS control media after 240 hrs. NF = normal dermal fibroblasts (n = 4), NS = normal dermal scar fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5), IKF = intra-lesional keloid fibroblasts (n = 5).
Figure 10.
Effect of exogenous TGFβ-1 on α-smooth muscle actin-(SMA) expression and cellular organisation.
A. Elevated α-SMA protein expression and cellular re-organisation into whirl-like structures were observed in PKF upon 24 hrs treatment with 1 ng/mL TGFβ-1. B. Elevated α-SMA expression and incorporation into microfilaments was observed in NF treated with 1 ng/mL TGFβ-1, although no cellular re-organisation was noted. Blue = nuclei, Green = α-SMA protein, NF = normal dermal fibroblasts (n = 4), PKF = peri-lesional keloid fibroblasts (n = 5).
Figure 11.
Proposed hypothetical mechanism of keloid recurrence.
A. H&E showing cross section of Keloid lesion including adjacent normal skin. IL: intra-lesional; PL: peri-leisonal compartments; Pap. Dermis: papillary dermis; Ret. Dermis: Reticular dermis. B. Keloid fibroblasts may be changed, for example by mechanical injury itself or as a consequence of the healing process eg inflammation, and exhibit epigenetic differences to normal fibroblasts that allow paracrine signalling to occur. This may result in upregulated fibrotic markers that influence increased collagen and fibronectin deposition in a self-sustaining manner. C. At the margins of the lesion, keloid fibroblasts influence (paracrine signaling) normal skin primary fibroblasts to upregulate fibrotic markers such as CTGF, PAI-1, α-SMA. CTGF is known mitogenic for fibroblasts and PAI-1 is known to increase collagen deposition through inhibiting PA activity. CTGF may increase both collagen and fibronectin concomitantly. PAI-1 may also influence cell migration by stimulating PA receptor and β3 integrin cycling by endocytosis. α-SMA may also influence cell tension and adhesion. Collectively these changes may influence migration into the surrounding healthy skin leading to high recurrence of keloid lesion, post-surgery.