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The pathogenesis and ideal treatment of keloid are still largely unknown, and it is essential to develop an objective assessment of keloid severity to evaluate the therapeutic response. We previously reported that our diffuse reflectance spectroscopy (DRS) system could assist clinicians in understanding the functional and structural condition of keloid scars. The purpose of this study was to understand clinical applicability of our DRS system on evaluating the scar severity and therapeutic response of keloid. We analyzed 228 spectral data from 71 subjects with keloid scars. The scars were classified into mild (0-3), moderate (4-7) and severe (8-11) according to the Vancouver scar scale. We found that as the severity of the scar increased, collagen concentration and water content increased, and the reduced scattering coefficient at 800 nm and oxygen saturation (SaO2) decreased. Using the DRS system, we found that collagen bundles aligned in a specific direction in keloid scars, but not in normal scars. Water content and SaO2 may be utilized as reliable parameters for evaluating the therapeutic response of keloid. In conclusion, the results obtained here suggest that the DRS has potential as an objective technique with which to evaluate keloid scar severity. In addition, it may be useful as a tool with which to track longitudinal response of scars in response to various therapeutic interventions.
© 2015 Optical Society of America
1. Introduction
Keloid scar is one result of abnormal wound healing. It grows beyond the original margins of the scar and does not regress with time. Pathologically, it is characterized by excessive accumulation of extracellular matrix, mostly type I collagen, in the dermis [1–3], and clinically, the diagnosis of keloid is usually made by the verification of the fact that the scar grows continuously and invasively beyond the borders of the original wound [1, 4]. The molecular mechanism of keloid pathogenesis remains unclear [5]. The treatment modalities include intralesional injection of corticosteroids or chemotherapy drugs (i.e., 5-fluorouracil or methotrexate), laser therapy, compression therapy, surgery, or radiation therapy [6–8]. However, there is no single satisfactory treatment for keloid.
For assessment of scar, many scales or tools have been developed during the last decades, but none appear ideal [9–11]. Most clinicians rely on clinical observation and palpation of the scar to monitor disease progression, but this is subjective and not quantitative. A wide range of non-invasive tools exist to monitor scars, including laser Doppler, ultrasonography, tonometry, two-photon excited fluorescence and second harmonic generation, and multi-photon microscopy [12–15]. However, the high instrument cost, the lack of objective procedures, or long duration measurements impede the wide application of these instruments in the clinical setting. Therefore, it is desirable to develop a less expensive tool that can assess the severity of scar efficiently and effectively in vivo.
Diffuse Reflectance Spectroscopy (DRS) is a non-invasive technique. A DRS system measures the characteristic diffuse reflectance spectrum of the tissue under investigation at visible to near-infrared wavelength range. Using a photon transport model and a least-squares curve-fitting algorithm, the absorption and scattering properties of samples can be deduced from the measured diffuse reflectance. Moreover, the absorption spectra derived from the DRS measurements can be further translated into the concentrations of tissue chromophores, such as hemoglobin, melanin, water and lipid. DRS has been employed to study the optical properties of various types of biological tissues, including brain and breast [16, 17]. Previously, we utilized our DRS system equipped with a diffusing probe to determine the optical properties of in vivo skin [18]. In addition, we performed a pilot study using our system to measure the optical properties and collagen concentration of keloid scars, normal scars and uninjured skin [19]. The Monte Carlo simulation results indicated the average interrogation depth of the diffusing probe was in the range from 293 to 949 μm within the 550 to 860 nm wavelength region [19].
The aim of this study was to assess the clinical application of the DRS system on keloid patients. We classified keloid scars as “mild”, “moderate” and “severe” according to the Vancouver scar scale (VSS). We evaluated the correlation between the four parameters derived from our DRS system, including collagen concentration, reduced scattering coefficient at 800 nm, oxygen saturation (SaO2), and water content, and the severity of keloid scars as well as the outcome of therapeutic response. Physiological meaning and implication of our measurement results will also be discussed in this paper.
2. Materials and methods
2.1 Diffuse reflectance spectroscopy (DRS) system
The system employed here for measuring the diffuse reflectance from skin is illustrated in Fig. 1. Briefly, a probe comprised of five multimode fibers with 440-μm core diameter and 0.22 numerical aperture was used for light delivery and collection. We used a Tungsten Halogen lamp (HL2000, Ocean Optics, FL) as a broadband light source, and a spectrometer (QE65000, Ocean Optics, FL) to measure the skin diffuse reflectance at wavelengths ranging from 500 to 1000 nm. This probe was equipped with a high scattering Spectralon slab (Labshpere, NH) for diffusing the light source efficiently, so that we could determine the optical properties of skin by a simple photon diffusion equation [19]. The Spectralon slab had a thickness of 2 mm, an index of refraction of 1.35, and μa and μs′ of about 10−5/mm and 50/mm at the wavelength of 660 nm, respectively [18]. The detection fiber penetrated the Spectralon layer and was flush with the lower surface of the Spectralon. There were four source fibers placed on the upper surface of the Spectralon and their distances from the detection fiber were; 1.44, 1.92, 2.4, and 2.88 mm, respectively. A 1 × 4 optical switch (Piezosystem Jena, Germany) was used to bridge the optical fiber connected to the broadband light source and one of the four source optical fibers at a time. The average time required to take one complete measurement was about 10 seconds. The results of preliminary in vivo studies obtained using this system have been described in detail elsewhere [18–20].
Fig. 1 Configuration of the diffuse reflectance spectroscopy (DRS) system and the diffusing probe. The spectrometer and the optical switch are connected to a laptop computer, and are coordinated and controlled by a graphic user interface developed based on LabVIEW software (National Instruments Corp., Austin, Texas). Abbreviations: CCD, charge-coupled device; MTL, modified two-layer; D, detector fiber; S, source fiber; P, parallel direction measurement; V, vertical direction measurement.
2.2 Modified two-layer model
The photon propagation model for the diffusing probe was derived from the two-layer photon diffusion model [21]. Model derivation is briefly described in the following. In a two-layer anisotropic medium system, the diffusion equation can be rewritten as:
where D = 1/3 (μa + μs’) is the diffusion constant, and Φ is the fluence rate. S is the source term, c is the speed of light in the medium, and i = 1, 2 is the number of the layer. Since the first layer is a high scattering material, the light source from the source fiber can be treated as a point source S1 = (x, y, z-z0) located in the first layer where z0 = 1/ (μa + μs’) and S2 = 0. By employing the extrapolated boundary condition that assumes the fluence and the flux are continuous at the boundary [22], the fluence rate of diffusion equation system can be solved in Fourier domain. In the modified two-layer geometry, the detector is located at the boundary of the first layer and the second layer. The fluence rate at the detector has the following form in the Fourier domain:where , , l is the thickness of the first layer, , and . Reff represents the fraction of photons that is internally diffusely reflected at the boundary, and Reff was determined to be 0.493 for n = 1.4, which represents the typical refractive index of skin. By performing inverse Fourier transform to the solution numerically, we can obtain the fluence rate at the detector. The spatially resolved reflectance can be calculated as the integral of the radiance L2 at the detector, where, over the backward hemisphere:where , and Rfres(θ) is the Fresnel reflection coefficient for a photon with an incident angle θ relative to the normal to the boundary [21].2.3 Measurement of scar by DRS System
Each measurement was taken at two sites; one at the active lesion of the keloid scar, and one at the uninjured skin site which was located 3 cm away from the border of the scar. All measurements were carried out in two orthogonal fiber alignment directions for each site (Fig. 1). This was achieved by rotating the diffusing probe by 90° after the first set of measurements was performed. The two measurement directions were defined as the optical fiber alignment of the diffusing probe parallel (P) or vertical (V) to the long axis of a scar. Each set of measurements was composed of 10 total measurements (5 measurements for P and others for V) in which the probe was physically removed and replaced each time.
Each set of skin reflectance spectra was first calibrated by the reflectance spectra measured from a siloxane phantom with known optical properties (μa = 0.07 mm−1 and μs’ = 1.91 mm−1 at 780 nm) to remove the instrument response. The calibrated spectra were then be reduced to an absorption spectrum and a reduced scattering spectrum. In practice, calibrated reflectance spectra were fit to the forward modified two-layer model derived above to obtain the absorption and scattering spectra of the tissue. To solve this inverse problem, the “lsqcurvefit” nonlinear curve fitting function in MATLAB® (MathWorks, MA, USA) was employed for performing the least-squares fittings. This algorithm is a subspace trust-region method and is based on the interior-reflective Newton method described in Ref [23]. The derived absorption spectrum μa(skin) can then be fit linearly to known chromophore absorption spectra, including oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water and collagen, to obtain their concentrations based on the following equation [19, 24–26]:
where C and ε represent the concentration and the extinction coefficient of a certain substance, respectively. It should be noted that although the absorption spectral trends of melanin and collagen are similar, they possess different slopes. In our previous study in which we recruited 12 subjects with keloids, it was found that the correlation coefficient of recovered melanin and collagen concentration was lower than 0.67 [19]. This suggests that Eq. (4) can be used to recover skin collagen concentration that would not be interfered by the presence of melanin. Moreover, as we considered the contribution of lipid to the skin absorption spectra in the chromophore fitting, we recovered values of almost zero or exact zero for all cases. We speculate that due to the shallow interrogation depths of our diffusing probe, most detected photons did not reach the subcutaneous layer. Thus, the contribution of lipid to the absorption spectra was not taken into account in Eq. (4). Besides, the derived reduced scattering spectra were fit to the classical Mie power law (μs' = a × λ-b) in the range 650 ~930 nm to smooth the raw scattering spectra.2.4 Keloid patients
In this study, 71 patients (35 males and 36 females) with keloid scars were recruited at National Cheng Kung University Hospital. The protocol was approved by the Institutional Review Board (No.: ER-100-332), and a written informed consent was obtained from all subjects prior to the measurements. The subjects ranged from 15 to 74 years of age with a spectrum of skin types from type III to type IV.
The clinical diagnosis of a normal scar was made if the height of the scar was < 2 mm and displayed uninjured skin color or hypopigmentation. The diagnosis of a keloid scar was based on identifying the erythematous criterion of the scar as well as verifying whether the scar had elevated and extended border that was beyond the original wound or lesion [27]. It has been reported that keloid and hypertrophic scars are pathologically different [1]. Clinically, we distinguished “hypertrophic scar” from “keloid” according to the scar classification schemes proposed by Mustoe et al. [4]. Specifically, keloids can be defined for the scars that grow continuously and invasively beyond the margin of the original wound, while hypertrophic scars are the scars that stay within the boundaries of the original wound and their growth rate regress with time. However, some scar lesions may simultaneously exhibit the characteristics of hypertrophic scar and keloid. Therefore, in this study, we did not recruit such kind of cases. The Vancouver scar scale (VSS) was rated by clinicians for each scar. The VSS evaluates the pliability, the height, the vascularity, and the pigmentation of the scar [28]. The DRS measurements and the evaluation of the VSS scores were conducted in a blinded fashion, i.e. the operator of the DRS system did not know the VSS score while measuring. The patients received intralesional corticosteroid injection (triamcinolone 10 mg/cc or 40 mg/cc) with or without additional dye laser therapy (7 mm, 12-14 J/cm2). They were treated at the frequency of every 4 to 6 weeks for a total time ranged from 3 to 11 months.
2.5 Histopathological studies
For the purpose of cosmetics and symptomatic relief, some patients received scar excision and subsequent radiotherapy according to the protocol proposed by Ogawa et al. [29]. The excised specimen of the scar was fixed in 10% formaldehyde, alcohol dehydrated, embedded in paraffin, cut into 4-mm sections, and stained with hematoxylin and eosin (H&E), Masson’s trichrome (MT) stain and sirius red staining for evaluation of collagen deposition [30]. The organization of collagen bundles were further examined by viewing Sirius red-stained histological sections with polarized light microscopy.
2.6 Statistical analysis
The experimental values were expressed as mean (AVG) ± standard error of the mean (SEM). The significance of difference between control and scar groups (e.g., normal scar or keloid) was assessed by paired Student’s t-test. The significance of difference between three severity groups was assessed by one-way analysis of variance followed by Bonferroni post-test using PRISM 5.0 (Graph Pad Software, La Jolla, CA). The significance of difference between parallel and vertical directions was assessed by paired Student’s t-test. The values of P<0.05, P<0.01 and P<0.001 were considered statistically significant and signified by the symbols “*”, “**” and “***”, respectively.
3. Results
3.1 The results of DRS system in keloid scars, normal scars and uninjured skin
Among the 71 patients recruited in this study, 24 patients had both keloid and normal scars. Figure 2(a) shows the representative clinical pictures of keloid (KS, black solid circle) and normal scars (NS, red solid circle) of one of such patients. The uninjured skin located 3 cm away from the keloid (CKS, black dashed circle) and normal scars (CNS, red dashed circle) were used as control groups. The VSS of keloid scars ranged from 3 to 11 points (mean 6.71 points), while that of normal scars ranged from 0 to 2 (mean 0.68 points). Figure 2(b) and Fig. 2(c) show the typical absorption and reduced scattering spectra of scar (solid lines) and uninjured skin (dashed lines), respectively. It can be seen in Fig. 2(b) and 2(c) that the absorption of keloid is higher than that of normal scars and uninjured skin in the 500 - 600 nm range and the magnitude of the reduced scattering spectrum of keloid is the lowest among all reduced scattering spectra. Chromophore fitting and scattering power law fitting were performed on the keloid absorption and reduced scattering spectra depicted in Fig. 2(b), and 2(c) and the results are demonstrated in Fig. 2(d) and 2(e), respectively. We can observe in Fig. 2(d) and 2(e) that the quality of chromophore fitting is in general well, while the raw reduced scattering spectra in the 500 - 600 nm range did not follow the trend predicted by the scattering power law. The absorption and reduced scattering spectra of the 24 patients were calculated and then further translated into the concentrations of collagen, oxygen saturation, and water content, as well as the reduced scattering coefficient at 800 nm. The results are summarized and plotted in Figs. 3(a)-3(e).
Fig. 2 (a) The clinical picture of a keloid patient containing keloid scar (KS), normal scar (NS). The uninjured skin 3-cm away from the border of keloid scars (CKS) or normal scars (CNS) was used as control groups. (b) Typical absorption, (c) reduced scattering spectra, (d) the chromophore fitting spectra, and (e) power law fitting of scars (solid lines), uninjured skin (dashed lines), and the results of fitting absorption spectra and reduced scattering spectra (up and down triangles).
Fig. 3 (a) Collagen concentration, (b) water content, (c) reduced scattering coefficient at 800 nm, (d) oxygen saturation (SaO2), and (e) two direction ratio of collagen concentration derived from 24 patients who had both keloid and normal scars. “P direction” (white) and “V direction” (black) represent the parallel and vertical alignment between probe and the long axis of scar lesions, respectively. The average of results of the P and V values is expressed by AVG. (gray). Data are shown as mean ± SEM. Statistical analysis was performed by paired Student’s t-test; * p<0.05, **p<0.01 and ***p<0.001.
For all sites under investigation, we performed measurements at two probe alignment directions. “P direction” and “V direction” represent the parallel and vertical alignment between probe and the long axis of scar lesions, respectively. In Fig. 3(a), the collagen concentration of NS was higher than CNS in the vertical direction (p < 0.05), and KS was higher than CKS in both parallel and vertical directions (both with p < 0.01). In addition, the collagen concentration of KS was higher than that of NS (parallel direction, p < 0.01; vertical direction, p < 0.001). Similar results can be observed for the water content in Fig. 3(b), where KS had higher water content than NS and uninjured skin. In contrast, the reduced scattering coefficient and oxygen saturation of KS were less than those of NS and uninjured skin (p < 0.001) (Figs. 3(c) and 3(d)). The two direction ratio of collagen concentration for KS, NS, and uninjured skin are shown in Fig. 3(e), which indicates that ratio for KS was significantly higher than CKS (p < 0.05). In contrast, the ratios for NS and CNS did not show significant difference. Besides, it can be noted that the ratio for uninjured skin was close to 1.
3.2 The results of DRS system in keloids of varying severities
We evaluated whether the DRS system can differentiate keloid scars of varying severities. We measured 135 lesions from 71 keloid patients using our DRS system. In total, we obtained 228 sets of data. These lesions were classified as mild scar (VSS 0-3, n = 69, mean = 1.46), moderate scar (VSS 4-7, n = 75, mean = 5.44) and severe scar (VSS 8-11, n = 84, mean = 9.155). Figure 4 shows the representative clinical pictures of keloid scars with increasing severities.
Fig. 4 Representative clinical pictures of keloid scars of different severities. The black circles indicate the measurement locations.
In order to reduce the variation associated with individual subjects and anatomical sites, the quantification was made by using a standardized ratio ([value of keloid scar – value of adjacent uninjured skin] / value of adjacent uninjured skin) of the four parameters. The results are shown in Fig. 5. It can be seen in Figs. 5(a)-5(d) that all four parameters recovered by our DRS system exhibited severity-dependent results. There was a significant difference (p<0.05, p < 0.01 or p < 0.001 by one-way analysis of variance with Bonferroni post-test) between mild and severe keloid scar in both the parallel and vertical measurement directions (Figs. 5(a)-5(d)). For the V/P ratios, there was no significant difference among keloids of all severities; however, the V/P ratios of keloids and uninjured skin demonstrated significant difference for all severity groups (Fig. 5(e)).
Fig. 5 DRS measurement results were translated into the standardized ratio, which was defined as ([value of keloid scar – value of adjacent uninjured skin] / value of adjacent uninjured skin). Data including (a) collagen concentration, (b) water content, (c) reduced scattering coefficient at 800 nm, (d) oxygen saturation (SaO2), and (e) two direction ratio of collagen concentration are shown as mean ± SEM. Statistical analysis was performed by one-way analysis of variance followed by Bonferroni post-test; *p<0.05, **p<0.01 and ***p<0.001.
3.3 The therapeutic effect of keloid lesions detected by DRS system
We then studied whether or not the DRS system was useful in monitoring changes of the keloid lesion induced by treatment. We recruited 20 patients who received local corticosteroid injections with or without pulsed dye laser treatment (585 nm, 1.5 ms pulse duration, 7 mm spot size and fluence 12-13.5 J/cm2). These 20 patients received regular treatment at the frequency of every 4 to 6 weeks. The DRS measurements were carried out during the first and the last visits of the patients. Subjects were separated into 2 groups, 9 with good response (the improvement of VSS score was equal or more than 3) and 11 with poor response (the improvement of VSS score was less than 3). Figure 6(a) and Fig. 6(b) demonstrate the representative clinical pictures of keloid lesions with good and poor responses, respectively.
Fig. 6 Four sets of representative clinical pictures of keloid with (a) good therapeutic response (case 1 and case 2), and (b) poor therapeutic response (case 3 and case 4).
Similar to the data processing method mentioned in the last subsection, standardized ratios were calculated for collagen concentration, water content, reduced scattering coefficients at 800 nm, and oxygen saturation and are displayed in Figs. 7 and 8. In the group of good responders, the water content decreased (P<0.05) while the SaO2 increased (P<0.05) after treatment (Figs. 7(b) and 7(d)). The collagen concentration, reduced scattering coefficient at 800 nm, and V/P value did not show a significant difference before and after treatment (Figs. 7(a), 7(c) and 7(e)). In contrast, there was no significant difference of the four parameters and V/P value before and after treatment in the group with poor response (Figs. 8(a)-8(e)).
Fig. 7 The standardized ratio, defined as ([value of keloid scar – value of adjacent uninjured skin] / value of adjacent uninjured skin), of (a) collagen concentration, (b) water content, (c) reduced scattering coefficient at 800 nm, (d) oxygen saturation, and (e) two direction ratio of collagen concentration for the group with good therapeutic response. Data are shown as mean ± SEM. Statistical analysis was performed by paired Student’s t-test; **p<0.01 and ***p<0.001.
Fig. 8 The standardized ratio, defined as ([value of keloid scar – value of adjacent uninjured skin] / value of adjacent uninjured skin), of (a) collagen concentration, (b) water content, (c) reduced scattering coefficient at 800 nm, (d) oxygen saturation, and (e) two direction ratio of collagen concentration for the group with poor therapeutic response. Data are shown as mean ± SEM.
4. Discussion
Scars are composed of extracellular matrix which mostly consists of collagen fibers, blood vessels, and fibroblasts in the dermis [2, 31]. In this study, collagen concentration, reduced scattering coefficient at 800 nm, SaO2, and water content percentage were chosen as important parameters to evaluate the severity of cutaneous scars. It is worth noting that since spatial heterogeneity exists within scars, The DRS measurement results of a scar site may not reflect the state of the whole scar tissue.
Most keloids have denser blood vessel networks than uninjured skin and their color can vary from pink or red to dark brown [2]. As shown in Fig. 2(b), the absorption of keloid is higher than that of normal scars and uninjured skin in wavelength range from 500 to 600 nm due to the strong absorption of hemoglobin. The absorption coefficient of keloids recovered in this wavelength region could reach as high as 0.8 mm−1. Besides, in this wavelength region, the reduced scattering spectra did not follow the scattering power law as shown in Fig. 2(e), and the reduced coefficients of keloids were less than 1 mm−1 in this region. Since the absorption and the reduced scattering coefficients had comparable values in 500-600 nm range, it can be inferred that the accuracy of the optical properties determined using the diffusion equation employed in this study could be poor in this region. However, all of the recovered absorption spectra still manifested absorption features of hemoglobin. We thus speculated that the absorption spectra might have incorrect absolute values in the 500-600 nm region, but their spectra shapes might be qualitatively similar to the true absorption spectra. We thus did not employ the concentrations of oxygenated hemoglobin or deoxygenated hemoglobin but only the oxygen saturation SaO2 in the data analyses as shown in Figs. 3, 5, 7, and 8. It is worth mentioning that, from the 75 patients we recruited, we found that the magnitude of absorption coefficients and SaO2 in the 500-600 nm region were always higher for keloid sites with redder color. Therefore, we believe that the determined SaO2 values were qualitatively meaningful. We are currently seeking for advanced photon transport models that can be utilized to recover the optical properties of skin from the diffuse reflectance with better accuracy.
The results shown in Figs. 3(a) and 5(a) indicate that our DRS system can differentiate the collagen concentration between keloid scars and normal scars from the same patient as well as quantify the severity of keloid lesions. Moreover, we noted that the collagen concentration measured in parallel and vertical directions exhibited different results, as shown in Fig. 3(a). It was speculated that this phenomenon was related to the alignment of collagen bundles in the keloid. After reviewing tissue sections of keloid specimens, we found that most of the collagen bundles were aligned in long fascicles parallel to the skin surface in tissue sections that were cut along the long axis of the keloid, while the collagen bundles tended to display a nodular morphology in the sections that were cut across the long axis of the keloid (Figs. 9(a) - (c)). In contrast, no such difference could be appreciated in the uninjured skin near the keloid lesion.
Fig. 9 Histopathological examinations revealed the different arrangement of collagen bundles between uninjured skin and keloid. (a) Representative examples of skin sections from keloid scar and uninjured skin tissues stained with hematoxylin and eosin (H&E). (b) The correspondent skin sections were stained with Masson trichrome (MT) stain. Collagen was stained blue, while the red signal was from non-collagenous extracellular matrix, blood vessels, arrector pili muscle and fibroblasts. (c) The keloid section with picrosirius red staining and imaged with polarized light microscopy. Bar = 200 μm.
We calculated the “two direction ratio” of the collagen concentrations and found that this ratio was close to unity for uninjured skin and otherwise for keloids as illustrated in Figs. 3(e) and 5(e). Based on the histopathlogical results shown in Fig. 9, it can be suggested that when this ratio was close to unity, the collagen distribution did not follow a certain direction; in contrast, the anisotropic distribution of collagen could result in the ratio deviating from unity. It can be found in Figs. 3(e) and 5(e) that the two direction ratio of collagen for keloids was higher than that of uninjured skin, which suggested the collagen distribution was more anisotropic in keloids than that of uninjured skin. However, as shown in Fig. 3(e), although the average value of the two direction ratio of collagen was higher for normal scars than for uninjured skin, there was no statistically significant difference between the two.
Keloids tend to occur at sites of high skin tension (e.g. anterior chest) and the collagen bundles within keloids are in general oriented parallel to the skin tension line [30, 32, 33]. Vehaegen et al. reported that keloid scars were organized in a more parallel manner than uninjured skin by calculating the width/length ratio of the scar images obtained using confocal microscopy [27]. To verify our DRS system’s capability to detect the anisotropy of collagen alignment, we performed measurements on a porcine Achilles tendon in parallel and vertical directions similar to the study of carried out by Kienle et al. [34]. The measurement results indicated the collagen concentration recovered in the vertical direction was higher than that in the parallel direction (p < 0.001 by paired Student’s t-test) (Fig. 10(b)). This result further supported that our DRS system can be used to differentiate various alignment conditions of collagen bundles. We infer that the V/P ratio of collagen concentration determined using our DRS system can represent the degree of anisotropy of the skin collagen network in which higher values represent a higher degree of anisotropy. The data illustrated in Figs. 3(e), 5(e), 7(e), and 8(e) show that the V/P ratio of keloids are always significant higher than that of uninjured skin. In addition, based on the pioneering work of Karl Langer [35], the principal direction of collagen orientation is along Langer’s lines, which may explain the V/P ratio of uninjured skin is close to 1, but not exactly equal to 1.
Fig. 10 (a) The vertical and parallel sections of a pig’s Achilles tendon from pig stained with H&E. (b) The collagen concentrations of a pig’s Achilles tendon from pig detected by DRS system in parallel and vertical directions. (*** p<0.001) Bar = 300 μm. Data are shown as mean ± SEM. Statistical analysis was performed by paired Student’s t-test.
It can be seen in Fig. 3(c) that the magnitude of the reduced scattering coefficient at 800 nm of keloid scars is smaller than that of uninjured skin, but a similar trend cannot be found for normal scars. In addition, it can be observed in Fig. 5(c) that the reduced scattering coefficient decreases with the severity of keloid. Furthermore, the reduced scattering coefficient was increased after treatment (P direction, p<0.05) in the group of good therapeutic response, as depicted in Fig. 7(c), but the results from the poor therapeutic responders did not show such trend (Fig. 8(c)). We previously conjectured that elastic fibers and collagen fibrils were responsible for the light scattering in the interrogation region of our diffusing probe [19]. Ishiko et al. reported that the elastic fibers was deficient in the extracellular matrix of keloids and the amount of elastic fibers could be recovered by successful treatment [36]. We speculate that our results shown in this paper are correlated with their finding.
Several research groups found that keloid tissues had higher water content than uninjured skin [37, 38]. Our results had led to a same conclusion in which we found that the water content was higher in keloid lesions (Fig. 3(b)), and the amount of water increased with the severity of keloid lesions (Fig. 5(b)). Interestingly, the water content was markedly reduced in the group of keloid with good therapeutic response but not in the group with poor therapeutic response as illustrated in Figs. 7(b) and 8(b), respectively. Although it is not statistically significant, the water content determined by our DRS system was sometimes orientation dependent. We suspect that this phenomenon which suggests inhomogeneous distribution of water could be related to the inhomogeneous collagen distribution in skin. Since collagen fibers possess the water-absorbing property, skin water homogeneity could be perturbed by the presence of collagen and spatial distribution of water could be related to the collagen organization.
The upregulation of plasminogen activator inhibitor-1 induced by hypoxia was identified as an important mechanism of keloid formation [39]. The data depicted in Fig. 3(d) and Fig. 5(d) showed that keloid tissues, especially in the severe keloids, are in hypoxic status compared with the uninjured skin and normal scars. In addition, the change of SaO2 level was dependent on the therapeutic response of keloid as illustrated in Figs. 7(d) and 8(d). It is noteworthy that we could not detect significant change of collagen concentration and V/P ratio in keloid group with good treatment response as shown in Figs. 7(a) and 7(e). This finding may be due to the long half-life of collagen degradation in keloid [40] and the short observation period (up to 11 months) in the present study.
The keloid patients recruited in the present study had Fitzpatrick skin type III and skin type IV. Although it has been reported that keloids are more common among patients with darker skin phototypes [41], we could not see the statistical significant difference between the uninjured skin melanin concentrations of keloidal and non-keloidal individuals. In addition, the correlation coefficient of recovered melanin and collagen concentration of the keloids was 0.331 in this study; this suggests that the melanin did not interfere in the collagen concentration recovery. Abnormal pigmentation has been noted within abnormal scars [42]. However, similar to the results shown in our previous study [19], we could not observe a statistical significant difference between the melanin concentrations of uninjured skin and keloid scars in this study (p = 0.707).
5. Conclusion
In summary, the results shown in this study demonstrate that the DRS system cannot only quantify the concentration of collagen, water content and oxygen saturation, but also determine the alignment direction of collagen bundles of keloid scars. In addition, the DRS system could differentiate the keloid lesions with differing severities, and the water content and oxygen saturation could be applied to monitor the therapeutic response of keloid. Our results obtained here suggest that the relatively low-cost DRS system has potential as an object technique with which to evaluate keloid scar severity. In addition, it may be useful as a tool with which to track longitudinal response of scars in response to various therapeutic interventions.
Acknowledgments
We appreciate Dr. Tai-Lan Tuan for her thorough review and constructive suggestions. This work was supported by National Science Council, Taiwan, under contracts NSC-102-2221-E-006-248-MY2 and NSC-102-2321-B-006-026.
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