Heat Shock Protein 70 Expression Patterns in Dermal Explants in Response to Ablative Fractional Phothothermolysis, Microneedle, or Scalpel Wounding

Abstract: Fractionated ablative laser intervention is effective for treating aged skin by inducing epidermal and/or dermal remodeling. The microneedle device is also used to initiate wound healing. In a previous study, the authors showed a clear time-dependent HSP70 expression profile subsequent to ablative fractional phothothermolysis (AFP) in a human skin explant model. The role of HSP70 after surgical interventions or microneedling is not fully understood. For this reason, the differences in spatio-temporal expressions of HSP70 by immunohistochemis-try in response to AFP, microneedling, and scalpel incisions were analyzed using a human skin explant model. Methods. AFP was performed using a scanned 250 µm CO₂-laser. The depth and density of the scalpel incisions were adapted to the defects reached by the laser or microneedle treatments. Skin explants were used as controls, for analysis of immediate responses, or they were subjected to cell culture medium for 1, 3, or 7 days. Results. HSP70 showed a clear time-dependent epidermal induction by AFP, peaking between 1 and 24 hours post-treatment with a significant decline within the following 7 days. HSP70 expression after scalpel incisions showed a similar time curve with less maximal HSP70 induction than in the laser-treated samples. Microneedling did not lead to histologically visible wounds. Conclusion. The human skin explant model was able to show a significant up-regulation of HSP70 after ablative thermal laser intervention, as well as surgical treatment, whereas microneedling did not reveal a significant up-regulation. This fact seems to be dependent on the amount of tissue wounding or stress.   Wound healing is a complex physiological process. Injury of the skin and concomitant blood vessel disruption lead to extravasation of blood constituents followed by platelet aggregation and blood clotting. These events initiate inflammation and set the stage for repair processes in which macrophages play a pivotal role. Re-epithelization begins within 24 hours post-injury, and matrix remodeling influences the tensile strength of the new tissue.¹ The remodeling process may take several weeks to many months to complete. The overlapping phases of wound healing, involving hemostasis, inflammation, angiogenesis, and resolution, are generally coordinated by multiple signal transmitters or growth factors, (eg, bradykinin, matrix metalloproteinases [MMP]), transforming growth factors [TGF], and heat shock proteins [HSP]).   Ablative skin resurfacing by a fractionated CO₂-laser or Er:YAG laser is a well-established treatment for the repair of photo-aged skin. These lasers remove the epidermal and parts of the dermal compartments depending on the amount of energy applied.² Using this technique, controlled collateral dermal heating is achieved next to microscopic ablation zones (MAZ). The spatially controlled thermal stress to the epidermis and the dermal compartments is followed by a wound healing response that ultimately leads to re-epithelization and dermal remodeling.^3–7 Prolonged erythema, edema, pigmentary alteration, and delayed wound healing are common adverse effects.⁸ For this reason, different non-ablative lasers and light sources have subsequently been developed for skin rejuvenation with the aim of reducing down time. Non-ablative procedures induce controlled, spatially determined, thermal damage, allowing subsequent collagen remodeling while preserving the epidermis.^9–14   The underlying molecular changes of both ablative and non-ablative remodeling are not fully understood as of yet, but dermal remodeling has been postulated to be induced by a time-dependent release of signal transducers, (eg, HSP70).^15–18   HSPs tend to be up-regulated in all cell types exposed to increasing temperatures (ie, 4˚C–6˚C above their physiologic temperature) or other forms of physical and chemical stress.^19–25 Therefore, HSPs are fundamentally involved in the protection of cells against damage, and play a role in cell reparation and wound healing.^26–28   The most important HSPs are the family members of HSP70 (HSP72 = 72 kDa and HSP73 = 73 kDa) and HSP47. Both HSP72 and HSP73 are present in the cytoplasm and the nucleus of keratinocytes, fibroblasts and adipocytes.^29,30 HSP73 is synthesized constitutively in all mammalian cells and is therefore often referred to as the “constitutive HSP70.” The synthesis of HSP72 is usually restricted to cells experiencing stress, and HSP72 is therefore often referred to as the “inducible HSP70.”²⁴ Several studies demonstrated an up-regulation of HSP70 in human keratinocytes by heat, UV-irradiation, wounding, inflammation, and different laser therapies around the “microscopic thermal injury zones” 1–48 hours post-treatment, leading ultimately to collagen remodeling.^{15,17,19,28,31–37} HSP47 expression is localized in the endoplasmatic reticulum of fibroblasts, where it is involved in the synthesis and transport of the pro-alpha1-(I)- and pro-alpha2-(I)-chains of procollagen I, leading to the formation of collagen I, III, and IV.^38–43 Up-regulation of HSP47 was detected in the skin beginning 7 days after skin wounding, and it lasted for more than 3 months.¹⁵   Incisional or scalpel wounds are characterized by clean wound margins and no substantial loss of epidermal or dermal structures. Above all, wound healing and scar formation depend on the correct combination of the wound margins. The microneedle device is offered as an alternative to fractional laser interventions, inducing a wound-healing cascade with collagen neogenesis and angiogenesis. It can be used for the treatment of acne scars, striae distensae, or skin remodeling. Due to the small needle diameter (0.1 mm), the microwounds close rapidly and the risk of infection is very low.   Different studies have compared wound healing after laser and surgical therapies and showed that lasers in general cause a delay in wound healing due to the collateral thermal damage.⁴⁴ However, no comparative studies have examined the influence and differences in HSP70 expression in these treatment options.   For this reason, the authors analyzed the spatio-temporal expressions of HSP70 in response to AFP, scalpel incisions, and microneedling in a skin explant model.

Methods

  Thirty-five skin samples obtained from routine skin surgery were used as skin explants. All subjects consented to the use of their skin explants. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in the approval by the institution’s Human Research Review Committee.   The explants were divided into three parts: one part (n = 15) was treated once with a fractionated CO₂ laser (10,600 nm, Exelo2, Quantel-Derma, Erlangen, Germany, spot size: 250 µm). The handpiece is designed to work like a scanner that can be adjusted to squares of various sizes (each axis can be 5, 10, 15, or 20 mm long (eg, 5 x 5, 5 x 10, 5 x 15, 5 x 20 mm; 10 x 5, 10 x 10, etc.). A treatment square is created spot after spot in a burst sequence by automatic movement of the scanner. Therefore, the pattern produced looks like a stamp, even though it is generated via scanning. The density can be chosen as 25/50/100/150/200/250/300/350/400 microspots per square centimeter. The distance between the center of the spot to the center of the next spot is: 2.11, 1.49, 1.05, 0.86, 0.75, 0.67, 0.61, 0.56, 0.53 mm. The ablative energies per microbeam (MB) adjusted to 50 mJ/MB, 100 mJ/MB, or 300 mJ/MB, result from various combinations of power and pulse duration (50 mJ/MB: 5 ms and 10 W; 100 mJ/MB: 5 ms and 20 W; 300 mJ/MB: 10 ms and 30 W. The density of the MAZ was set to 200, 150, or 100 per square centimeter.   The second part was incised with a scalpel (n = 15), whereas the third part was treated with a microneedle device (n = 5, [Dermaroller CIT8®, Dermasence, Germany]). The microneedle device has 24 circular arrays of 8 needles each (total needles: 192) in a cylindrical assembly with a 2-cm cylinder diameter and 2-cm length. The microneedle device was rolled five times in four directions, inducing approximately 250 microwounds/cm₂. The microneedles had a needle length of 1.5 mm. The depth and density of the scalpel incisions were adapted to the defects reached by the laser or microneedle treatments.   Routine pathology. One aliquot of the explants was fixed in 4% buffered formalin immediately after laser, microneedle and scalpel procedures, whereas the remaining aliquots were subjected to cell culture medium (DMEM enriched with streptavidine and 10% fetal calf serum) for 1, 3, or 7 days at a constant temperature of 31˚C–32˚C corresponding to an average skin surface temperature. Another aliquot of the explant was fixed in 4% buffered formalin without the preceding procedure to serve as a baseline control (neat). Following formalin fixation, all skin explants were embedded in paraffin, sectioned into 4 µm–6 µm thick slices, and stained with hematoxylin and eosin (H&E) according to the institution’s routine protocol.   Immunohistochemistry. The sections were incubated with 0.5% BSA/PBS (phosphate buffered saline/bovine serum albumin: 0.5% BSA prepared in PBS) for 5–10 minutes at 41˚C–43˚C, washed two times for 10 minutes each in PBS, and air dried. Afterward, the sections were incubated in a wet chamber for another 30 minutes at room temperature. The reaction was stopped with 25 µL–50 µL of normal sheep serum, and the slides were washed two times for 10 minutes each in PBS. The primary antibody anti-HSP70 (1:1 PBS/0.1% Tween), specific for HSP70, recognizes both the constitutive (HSP73) and inducible (HSP72) forms of HSP70. It was added to the sections and incubated in a wet chamber for 45 minutes at 37˚C. In order to visualize mAb binding, the Dako Real Detection System (K5005, Dako, Hamburg, Germany, Alkaline Phosphatase/RED, Rabbit/Mouse) was used according to the manufacturer’s protocol.   TGF-β was injected intradermally using a super fine syringe at concentrations of 5 ng/µL (20 µL or 40 µL) into control skin samples (positive controls) based on the investigations of Ong et al.⁴⁵   Evaluation of the staining intensity. All tissue samples were stained at the same time using identical procedures. HSP70 expression was analyzed microscopically by two independent investigators using different magnifications (1.25, 4, 10, 20, 40, 60 [Olympus BX41]) and docu-mented using a calibrated digital camera system (Olympus DP72) together with the software evaluation package (Olympus Cell F). The expression densities of HSP70 in skin explants ranged from 0 = undetected, 1 = low density, 2 = medium density, 3 = dense, and 4 = very dense, as Souil et al¹⁷ have described previously.

Statistical Analysis

  Data analyses were performed using non-parametric tests (Kruskal-Wallis ANOVA) and median tests (multiple comparisons, 2-tailed) as appropriate for data type and distribution (investigated with the Shapiro-Wilk W test or Kolmogorov-Smirnov test) to evaluate statistical signifi-cance (P < 0.05). Calculations were performed with the statistical computer program STATISTICA 7.0 (StatSoft, Inc., Tulsa, OK). P < 0.05 was considered statistically significant. All values are expressed as mean ± SEM.

Results

  Out of 180 aliquots of 40 fresh human skin explants, 60 were immediately subjected to ablative fractional laser intervention after surgery, and another 60 were subjected to scalpel incision. Twenty aliquots were treated with the microneedle device to measure the overall expression of HSP70 over 7 days. Forty aliquots served as non-treated controls. Skin samples were obtained from the trunk (n = 18, 51.4%), arms (n = 12, 34.2%), and legs (n = 5, 14.3%). Patient ages ranged from 42 to 72 years (mean 59.9 years ± 11.2). Twenty-three of the 35 patients (65.7%) were male and 12 (34.3%) were female. The original localization of skin explants, as well as the age of the donors, did not influence HSP70 expression.   As expected, increasing energy levels using paired combinations of pulse duration and power increased the dimensions of the resulting MAZ.   Overall, the highest constitutive expression of HSP70 after laser and surgical intervention was observed in the epidermal compartment, with no substantial differences between the spinocellular and basocellular layer. Expression was minor within the dermal papillary layer, around sebaceous glands, hair follicles, and blood vessels. HSP70 expression was very weak or absent within the stratum corneum, fibroblasts, and adipose tissue.   AFP resulted in a homogeneous up-regulation of HSP70 expression in the epidermal layers between the MAZ in the different treatment groups immediately (approximately 60 minutes) after AFP, and lasted up to 24 hours after the laser procedure with subsequent decline over the following 7 days (Figures 1 and 3). The HSP70 intensities post-AFP of all energy groups showed significant up-regulations between the time points neat and day 0 (P < 0.02), day 1 (P < 0.01), or day 3 (P = 0.02), and down-regulations from day 0 (P < 0.03) or day 1 (P < 0.01) to day 7 without clear differences in HSP70 expression between the different treatment groups. Significant differences in HSP70 expression over time could not be demonstrated within the different treatment groups because of the small sample numbers.   HSP70 expression after scalpel incisions showed a similar time curve with diminished HSP70 induction than in the laser-treated samples (Figures 2 and 3). The HSP70 intensities in scalpel-incised explants showed a significant up-regulation between the time points neat and day 0 (P = 0.02), and a down-regulation from day 0 to day 7 (P = 0.01).   Wounding was not visible histologically after the use of the microneedle device, and the intensities of the microneedle-induced HSP70 expression levels did not clearly differ among time points.

Discussion

  The ban on animal testing in the European cosmetic industry has increased the urgency to develop innovative alternative skin models to replace the use of laboratory animals.^46–48 Various skin models are now available; however, they often lack dermal compartments, and only a few models have been validated by relevant regulatory authorities, such as the European Center for Validation of Alternative Methods (ECVAM).^49,50 In a previous study, the authors showed a clear time-dependent HSP70 expression profile post-AFP using a scanned 250 µm CO₂ laser beam in a human skin explant model.   Several studies have compared wound healing after laser and surgical treatment options. The results differ largely, although most of the studies showed that laser therapies cause a delay in wound healing probably due to the collateral thermal damage.⁴⁴ Histological evaluation re-vealed that re-epithelialization is complete in excisional wounds by day 5, whereas laser wound resurfacing is not complete until day 7.⁵¹ The adjacent hyperproliferative epidermis is slightly more pronounced in laser wounds. Besides, the increased synthesis of HSP70 due to thermal laser interventions is generally accompanied by a reduction in protein synthesis, causing a temporary cessation of cell proliferation that is eventually relevant for this delayed wound healing.¹⁷ Consequently, researchers have attempted to modify this thermal injury and thus reduce wound healing delays.⁵² In contrast, in line with observations in disease conditions resulting in delayed wound healing, wound healing was improved in diabetic rats by local treatment with a non-toxic HSP70 inducer (bimoclomol).⁵³ Studies measuring the tissue tensile strength after different laser and surgical interventions demonstrated controversial results,^52,54 as did studies examining the extent and influence of inflammation and edema.^54,55 It is possible that thermal injury and necrosis prolong the inflammatory phase, resulting in sustained levels of MMPs. To the best of the authors’ knowledge, no studies have examined histological changes after microneedling, and none of the studies comparing laser- and surgically-induced wounds examined the differences in HSP70 expression and its value for the wound healing process. For this reason, the spatio-temporal expression levels of HSP70 in response to AFP, scalpel incisions, and microneedling were examined.   HSP70 assists in protein folding and allows the functional state of proteins to be maintained under conditions, such as physical or chemical stress, as well as different laser therapies.^{15,17,19,22,23,28,32–37,56} HSP70 also plays a role in inducing the expression of growth factors, such as TGF-β, which is a key element in wound healing and fibrogenic processes.^16,57–59 However, TGF-β also induces HSP70 heat independently.⁶⁰ The authors took advantage of this phenomenon as a positive control for the induction of HSP70 heat and stress independently.   Consequently, it was observed that significant induction of HSP70 began 1 hour post-AFP with maximal expression levels at 24 hours and a subsequent decline over the following week in the skin explant model. As expected, mean lesion dimensions increased with increasing energy used. Maximum HSP70 expression was detected in epidermal compartments between the MAZ and the surrounding tissue of “microscopic thermal injury zones,” paralleling results reported by others^22,35,36 studying both HSP70 forms. In contrast, Hantash et al15 detected the expression of HSP72 (the inducible form of HSP70) as early as 2 days post-ablative fractional re-surfacing, and the expression diminished significantly over 3 months (spot size: 120 µm, 30 W, 5–40 mJ). Previously, the authors showed that Er:glass laser intervention led to an up-regulation of HSP70 with the maximum at 1 to 3 days post-intervention.   After surgical incisions, a similar but lower course of HSP70 up-regulation was observed, probably due to the lack of a thermal effect. This finding corresponds with examinations in which skin cooling using a sapphire cooling plate attached to the handpiece of an 1319 nm Nd:YAG laser only led to significant HSP70 expression in rat skin at 13˚C or warmer, whereas no identifiable cellular reaction was observed when the cooling was less than 5˚C.³⁷ However, for consistent and reliable HSP70 expression, high-temperature, shorter exposure protocols (9.17 mJ/cm2, 60–150 seconds) were less efficient than low-temperature, longer duration protocols (7.64 mJ/cm2).⁶¹ The differences between the laser-, scalpel-, or microneedling-induced HSP70 expression levels were not significant at any time point in the authors’ skin explant model. Microneedling did not induce microscopically visible wounds and did not up-regulate the HSP70 expression.

Conclusion

  The human skin explant model was able to show a significant up-regulation of HSP70 after ablative thermal laser intervention, as well as surgical treatment. Microneedling did not reveal significant HSP70 up-regulation, which seems to be dependent on the amount of tissue wounding or stress. The authors are from the University of Leipzig, Department of Dermatology, Allergology, and Venerology, Leipzig, Germany Address correspondence to: Doris Helbig, MD University of Leipzig, Department for Dermatology, Allergology, and Venerology Philipp-Rosenthal-Str. 23 04103 Leipzig Germany Phone: 0049-3419718611 Email: doris.helbig@yahoo.de

References

1. Clark RA. Cutaneous tissue repair: basic biologic considerations. I. J Am Acad Dermatol. 1985;13(5 Pt 1):701–725. 2. Tannous Z. Fractional resurfacing. Clin Dermatol. 2007;25(5):480–486. 3. Ross EV, Naseef GS, McKinlay JR, et al. Comparison of carbon dioxide laser, erbium:YAG laser, dermabrasion, and dermatome: A study of thermal damage, wound contraction, and wound healing in a live pig model: Implications for skin resurfacing. J Am Acad Dermatol. 2000;42(1 Pt 1):92–105. 4. Hruza GJ, Dover JS. Laser skin resurfacing. Arch Dermatol. 1996;132(4):451–455. 5. Anvari B, Milner TE, Tanenbaum BS, Kimel S, Svaasand LO, Nelson JS. Selective cooling of biological tissues: application for thermally mediated therapeutic procedures. Phys Med Biol. 1995;40(2):241–252. 6. Kelly KM, Majaron B, Nelson JS. Nonablative laser and light rejuvenation: the newest approach to photodamaged skin. Arch Facial Plast Surg. 2001;3(4):230–235. 7. Schmults CD, Phelps R, Goldberg DJ. Nonablative facial remodeling: erythema reduction and histologic evidence of new collagen formation using a 300-microsecond 1064-nm Nd:YAG laser. Arch Dermatol. 2004;140(11):1373–1376. 8. Nanni CA, Alster TS. Complications of carbon dioxide laser resurfacing. An evaluation of 500 patients. Dermatol Surg. 1998;24(3):315–320. 9. Fournier N, Lagarde JM, Turlier V, Courrech L, Mordon S. A 35-month profilometric and clinical evaluation of non-ablative remodeling using a 1540-nm Er:glass laser. J Cosmet Laser Ther. 2004;6(3):126–130. 10. Fournier N, Mordon S. Nonablative remodeling with a 1,540 nm erbium:glass laser. Dermatol Surg. 2005;31(9 Pt 2):1227–1235. 11. Mordon S, Capon A, Creusy C, et al. In vivo experimental evaluation of skin remodeling by using an Er:Glass laser with contact cooling. Lasers Surg Med. 2000;27(1):1–9. 12. Fournier N, Dahan S, Barneon G, et al. Nonablative remodeling: a 14-month clinical ultrasound imaging and profilometric evaluation of a 1540 nm Er:Glass laser. Dermatol Surg. 2002;28(10):926–931. 13. Lupton JR, Williams CM, Alster TS. Nonablative laser skin resurfacing using a 1540 nm erbium glass laser: a clinical and histologic analysis. Dermatol Surg. 2002;28(9):833–835. 14. Dahan S, Lagarde JM, Turlier V, Courrech L, Mordon S. Treatment of neck lines and forehead rhytids with a nonablative 1540-nm Er:glass laser: a controlled clinical study combined with the measurement of the thickness and the mechanical properties of the skin. Dermatol Surg. 2004;30(6):872–880. 15. Hantash BM, Bedi VP, Kapadia B, et al. In vivo histological evaluation of a novel ablative fractional resurfacing device. Lasers Surg Med. 2007;39(2):96–107. 16. Arany PR, Nayak RS, Hallikerimath S, Limaye AM, Kale AD, Kondaiah P. Activation of latent TGF-beta1 by low-power laser in vitro correlates with increased TGF-beta1 levels in laser-enhanced oral wound healing. Wound Repair Regen. 2007;15(6):866–874. 17. Souil E, Capon A, Mordon S, Dinh-Xuan AT, Polla BS, Bachelet M. Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin. Br J Dermatol. 2001;144(2):260–266. 18. Fatemi A, Weiss MA, Weiss RA. Short-term histologic effects of nonablative resurfacing: results with a dynamically cooled millisecond-domain 1320 nm Nd:YAG laser. Dermatol Surg. 2002;28(2):172–176. 19. Zhou JD, Luo CQ, Xie HQ, et al. Increased expression of heat shock protein 70 and heat shock factor 1 in chronic dermal ulcer tissues treated with laser-aided therapy. Chin Med J (Engl) . 2008;121(14):1269–1273. 20. O’Connell-Rodwell CE, Shriver D, Simanovskii DM, et al. A genetic reporter of thermal stress defines physiologic zones over a defined temperature range. FASEB J. 2004;18(2):264–271. 21. Nagata K, Hirayoshi K, Obara M, Saga S, Yamada KM. Biosynthesis of a novel transformation-sensitive heat-shock protein that binds to collagen. Regulation by mRNA levels and in vitro synthesis of a functional precursor. J Biol Chem. 1988;263(17):8344–8349. 22. Blake MJ, Gershon D, Fargnoli J, Holbrook NJ. Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats. J Biol Chem. 1990;265(25):15275–15279. 23. Maytin EV, Wimberly JM, Anderson RR. Thermotolerance and the heat shock response in normal human keratinocytes in culture. J Invest Dermatol. 1990;95(6):635–642. 24. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 1992;72(4):1063–1081. 25. Morimoto RI. Cells in stress: transcriptional activation of heat shock genes. Science. 1993;259(5100):1409–1410. 26. Simon MM, Reikerstorfer A, Schwarz A, et al. Heat shock protein 70 overexpression affects the response to ultraviolet light in murine fibroblasts. Evidence for increased cell viability and suppression of cytokine release. J Clin Invest. 1995;95(3):926–933. 27. Ravagnan L, Gurbuxani S, Susin SA, et al. Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat Cell Biol. 2001;3(9):839–843. 28. Laplante AF, Moulin V, Auger FA, et al. Expression of heat shock proteins in mouse skin during wound healing. J Histochem Cytochem. 1998;46(11):1291–1301. 29. Kiang JG, Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther. 1998;80(2):183–201. 30. Beckham JT, Mackanos MA, Crooke C, et al. Assessment of cellular response to thermal laser injury through bioluminescence imaging of heat shock protein 70. Photochem Photobiol. 2004;79(1):76–85. 31. Blake D, Svalander P, Jin M, Silversand C, Hamberger L. Protein supplementation of human IVF culture media. J Assist Reprod Genet. 2002;19(3):137–143. 32. Trautinger F, Trautinger I, Kindås-Mügge I , Metze D, Luger TA. Human keratinocytes in vivo and in vitro constitutively express the 72-kD heat shock protein. J Invest Dermatol. 1993;101(3):334–338. 33. Trautinger F, Kindås-Mügge I, Barlan B, Neuner P, Knobler RM. 72-kD heat shock protein is a mediator of resistance to ultraviolet B light. J Invest Dermatol. 1995;105(2):160–162. 34. Capon A, Souil E, Gauthier B, et al. Laser assisted skin closure (LASC) by using a 815-nm diode-laser system accelerates and improves wound healing. Lasers Surg Med. 2001;28(2):168–175. 35. Laubach HJ, Tannous Z, Anderson RR, Manstein D. Skin responses to fractional photothermolysis. Lasers Surg Med. 2006;38(2):142–149. 36. Brown SA, Farkas JP, Arnold C, et al. Heat shock proteins 47 and 70 expression in rodent skin model as a function of contact cooling temperature: Are we overcooling our target? Lasers Surg Med. 2007;39(6):504–512. 37. Hantash BM, Bedi VP, Chan KF, Zachary CB. Ex vivo histological characterization of a novel ablative fractional resurfacing device. Lasers Surg Med. 2007;39(2):87–95. 38. Takechi H, Hirayoshi K, Nakai A, Kudo H, Saga S, Nagata K. Molecular cloning of a mouse 47-kDa heat-shock protein (HSP47), a collagen-binding stress protein, and its expression during the differentiation of F9 teratocarcinoma cells. Eur J Biochem. 1992;206(2):323–329. 39. Nagata K, Saga S, Yamada KM. A major collagen-binding protein of chick embryo fibroblasts is a novel heat shock protein. J Cell Biol. 1986;103(1):223–229. 40. Nagata K. HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development. Semin Cell Dev Biol. 2003;14(5):275–282. 41. Nagai N, Hosokawa M, Itohara S, et al. Embryonic lethality of molecular chaperone hsp47 knockout mice is associated with defects in collagen biosynthesis. J Cell Biol. 2000;150(6):1499–1506. 42. Nagata K. Expression and function of heat shock protein 47: a collagen-specific molecular chaperone in the endoplasmic reticulum. Matrix Biol. 1998;16(7):379–386. 43. Ishida Y, Kubota H, Yamamoto A, et al. Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis. Mol Biol Cell. 2006;17(5):2346–2355. 44. Molgat YM, Pollack SV, Hurwitz JJ, et al. Comparative study of wound healing in porcine skin with CO2 laser and other surgical modalities: preliminary findings. Int J Dermatol. 1995;34(1):42–47. 45. Ong SE, Blagoev B, Kratchmarova I, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002(5);1:376–386. 46. Festing S, Wilkinson R. The ethics of animal research. Talking Point on the use of animals in scientific research. EMBO Rep. 2007;8(6):526–530. 47. Halder M. Three Rs potential in the development and quality control of immunobiologicals. ALTEX. 2001;18(Suppl 1):13–47. 48. Hendriksen CF. Refinement, reduction, and replacement of animal use for regulatory testing: current best scientific practices for the evaluation of safety and potency of biologicals. ILAR J. 2002;43(Suppl):S43–S48. 49. Netzlaff F, Lehr CM, Wertz PW, Schaefer UF. The human epidermis models EpiSkin, SkinEthic and EpiDerm: an evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport. Eur J Pharm Biopharm. 2005;60(2):167–178. 50. Ponec M. Skin constructs for replacement of skin tissues for in vitro testing. Adv Drug Deliv Rev. 2002;54(Suppl 1):S19–S30. 51. Draper BK, Davidson MK, Nanney LB. MMPs and TIMP-1 are differentially expressed between acute murine excisional and laser wounds. Lasers Surg Med. 2002;30(2):106–116. 52. Sanders DL, Reinisch L. Wound healing and collagen thermal damage in 7.5-microsec pulsed CO(2) laser skin incisions. Lasers Surg Med. 2000;26(1):22–32. 53. Vigh L, Literati PN, Horvath I, et al. Bimoclomol: a nontoxic, hydroxylamine derivative with stress protein-inducing activity and cytoprotective effects. Nat Med. 1997;3(10):1150–1154. 54. Wu N, Jansen ED, Davidson JM. Comparison of mouse matrix metalloproteinase 13 expression in free-electron laser and scalpel incisions during wound healing. J Invest Dermatol. 2003;121(4):926–932. 55. Bellina JH, Hemmings R, Voros JI, Ross LF. Carbon dioxide laser and electrosurgical wound study with an animal model: a comparison of tissue damage and healing patterns in peritoneal tissue. Am J Obstet Gynecol. 1984;148(3):327–334. 56. Charveron M, Calvo M, Gall Y. Cell stress and implications of the heat-shock response in skin. Cell Biol Toxicol. 1995;11(3-4):161–165. 57. Kilani RT, Guilbert L, Lin X, Ghahary A. Keratinocyte conditioned medium abrogates the modulatory effects of IGF-1 and TGF-beta1 on collagenase expression in dermal fibroblasts. Wound Repair Regen. 2007;15(2):236–244. 58. Munger JS, Huang X, Kawakatsu H, et al. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 1999;96(3):319–328. 59. Barcellos-Hoff MH, Dix TA. Redox-mediated activation of latent transforming growth factor-beta 1. Mol Endocrinol. 1996;10(9):1077–1083. 60. Takenaka IM, Hightower LE. Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells. J Cell Physiol. 1992;152(3):568–577. 61. Wilmink GJ, Opalenik SR, Beckham JT, et al. Molecular imaging-assisted optimization of hsp70 expression during laser-induced thermal preconditioning for wound repair enhancement. J Invest Dermatol. 2009;129(1):205–216.