Implications for cumulative and prolonged clinical improvement induced by cross-linked hyaluronic acid: An in vivo biochemical/microscopic study in humans.

In photoaged human skin, type I collagen fragmentation impairs dermal extracellular matrix (ECM) integrity, resulting in collapsed/contracted fibroblasts with reduced type I procollagen synthesis. Injections of cross-linked hyaluronic acid (CL-HA) reverse these deleterious changes. To investigate the time course and effects of biochemical changes induced by injected CL-HA, particularly whether fibroblast activation leads to accumulation/deposition of dermal collagen, we injected CL-HA into photoaged skin of human participants over 60 years-old and performed biochemical/microscopic analyses of skin samples. Beginning 1 week post-injection and lasting 6-9 months, fibroblasts exhibited activation, including increased immunostaining and gene expression of markers of type I collagen synthesis, such as heat shock protein 47 and components of the transforming growth factor-ß pathway. At 1 week post-injection, multiphoton microscopy revealed elongation/stretching of fibroblasts, indicating enhanced dermal mechanical support. At 4 weeks, second-harmonic generation microscopy revealed thick collagen bundles densely packed around pools of injected CL-HA. At 12 months, accumulation of thick collagen bundles was observed and injected CL-HA remained present in substantial amounts. Thus, by occupying space in the dermal ECM, injected CL-HA rapidly and durably enhances mechanical support, stimulating fibroblast elongation and activation, which results in thick, densely packed type I collagen bundles accumulating as early as 4 weeks post-injection and continuing for at least a year. These observations indicate that early and prolonged clinical improvement following CL-HA injection results from space-filling and collagen deposition. As type I collagen has an estimated half-life of 15 years, our data provide the foundations for optimizing the timing/frequency of repeat CL-HA injections.

Cutaneous Hypervascularization Treatment Using Photo-Mediated Ultrasound Therapy.

Photo-mediated ultrasound therapy (PUT) is a cavitation-based, highly selective antivascular technique. In this study, the effectiveness and safety of PUT on cutaneous vascular malformation was examined through in vivo experiments in a clinically relevant chicken wattle model, whose microanatomy is similar to that of port-wine stain and other hypervascular dermal diseases in humans. Assessed by optical coherence tomography angiography, the blood vessel density in the chicken wattle decreased by 73.23% after one session of PUT treatment in which 0.707 J/cm2 fluence laser pulses were applied concurrently with ultrasound bursts (n = 7, P < .01). The effectiveness of removing blood vessels in the skin at depth up to 1 mm was further assessed by H&E-stained histology at multiple time points, which included days 1, 3, 7, 14, and 21 after treatment. Additional immunohistochemical analyses with CD31, caspase-3, and Masson's trichrome stains were performed on day 3 after treatment. The results show that the PUT-induced therapeutic effect was confined and specific to blood vessels only, whereas unwanted collateral damage in other skin tissues such as collagen was avoided. The findings from this study demonstrate that PUT can efficiently and safely remove hypervascular dermal capillaries using laser fluence at a level that is orders of magnitude smaller than that used in conventional laser treatment of vascular lesions, thus offering a safer alternative technique for clinical management of cutaneous vascular malformations.

Needs and Gaps in Resident Trainee Education, Clinical Patient Care, and Clinical Research in Cosmetic Dermatology: Position Statement of the Association of Academic Cosmetic Dermatology.

Cosmetic dermatology is a key subspecialty of academic dermatology. As such, academic centers are expected to demonstrate excellence in the teaching of cosmetic dermatology skills to trainees, the clinical delivery of cosmetic dermatology services to patients, and the performance of clinical research that advances knowledge and uncovers new therapies in cosmetic dermatology. The Association of Academic Cosmetic Dermatology (AACD), a newly formed medical professional society, includes as its principal aims the support of all of these areas. AACD is comprised of group of board-certified dermatologists who teach cosmetic and laser dermatology at US dermatology residency programs. An expert panel constituted by the AACD recently convened a workshop to review gaps pertaining to academic cosmetic dermatology. This panel considered needs and potential corrective initiatives in three domains: resident education, patient experience, and clinical research. The work of the panel was used to develop a roadmap, which was adopted by consensus, and which will serve to guide the AACD moving forward.

The Association of Academic Cosmetic Dermatology: improving cosmetic dermatology education through collaboration, research, and advocacy.

Cosmetic and laser procedures are increasingly popular among patients and are skills in which dermatologists are regarded as well trained. Most dermatology residents intend to incorporate cosmetic procedures into their practice and prefer to learn such procedures during residency through direct patient care. However, there are notable challenges in optimizing how residents are trained in cosmetic and laser dermatology. To address these barriers and elevate the practice of cosmetic dermatology in academic medicine, the Association of Academic Cosmetic Dermatology (AACD) was founded in 2021 as the lead professional society for dermatologists who direct the education of resident trainees in cosmetic and laser dermatology. The AACD, a group of board-certified dermatologists who teach cosmetic and laser dermatology to residents, aims to improve cosmetic dermatology education through collaboration, research, and advocacy.

Evidence-Based Clinical Practice Guidelines for Laser-Assisted Drug Delivery.

Importance: Laser-assisted drug delivery (LADD) is used for various medical and cosmetic applications. However, there is insufficient evidence-based guidance to assist clinicians performing LADD. Objective: To develop recommendations for the safe and effective use of LADD. Evidence Review: A systematic literature review of Cochrane Central Register of Controlled Trials, Embase, and MEDLINE was conducted in December 2019 to identify publications reporting research on LADD. A multidisciplinary panel was convened to draft recommendations informed by the systematic review; they were refined through 2 rounds of Delphi survey, 2 consensus meetings, and iterative review by all panelists until unanimous consensus was achieved. Findings: Of the 48 published studies of ablative fractional LADD that met inclusion criteria, 4 were cosmetic studies; 21, oncologic; and 23, medical (not cosmetic/oncologic), and 6 publications of nonablative fractional LADD were included at the request of the expert panel, producing a total of 54 studies. Thirty-four studies (63.0%) were deemed to have low risk of bias, 17 studies (31.5%) had moderate risk, and 3 (5.5%) had serious risk. The key findings that informed the guidelines developed by the expert panel were as follows: LADD is safe in adults and adolescents (≥12 years) with all Fitzpatrick skin types and in patients with immunosuppression; it is an effective treatment for actinic keratosis, cutaneous squamous cell carcinoma in situ, actinic cheilitis, hypertrophic scars, and keloids; it is useful for epidermal and dermal analgesia; drug delivery may be increased through the application of heat, pressure, or occlusion, or by using an aqueous drug solution; laser settings should be selected to ensure that channel diameter is greater than the delivered molecule; antibiotic prophylaxis is not recommended, except with impaired wound healing; antiviral prophylaxis is recommended when treating the face and genitalia; and antifungal prophylaxis is not recommended. The guideline's 15 recommendations address 5 areas of LADD use: (I) indications and contraindications; (II) parameters to report; (III) optimization of drug delivery; (IV) safety considerations; and (V) prophylaxis for bacterial, viral, and fungal infections. Conclusions and Relevance: This systematic review and Delphi consensus approach culminated in an evidence-based clinical practice guideline for safe and effective use of LADD in a variety of applications. Future research will further improve our understanding of this novel treatment technique.

Removing Subcutaneous Microvessels Using Photo-Mediated Ultrasound Therapy.

BACKGROUND AND OBJECTIVES: We have developed a novel anti-vascular technique, termed photo-mediated ultrasound therapy (PUT), which utilizes nanosecond duration laser pulses synchronized with ultrasound bursts to remove the microvasculature through cavitation. The objective of the current study is to explore the potential of PUT in removing subcutaneous microvessels. STUDY DESIGN/MATERIALS AND METHODS: The auricular blood vessels of two New Zealand white rabbits were treated by PUT with a peak negative ultrasound pressure of 0.45 MPa at 0.5 MHz, and a laser fluence of 0.056 J/cm2 at 1064 nm for 10 minutes. Blood perfusion in the treated area was measured by a commercial laser speckle imaging (LSI) system before and immediately after treatment, as well as at 1 hour, 3 days, 2 weeks, and 4 weeks post-treatment. Perfusion rates of 38 individual vessels from four rabbit ears were tracked during this time period for longitudinal assessment. RESULTS: The measured perfusion rates of the vessels in the treated areas, as quantified by the relative change in perfusion rate, showed a statistically significant decrease for all time points post-treatment (P < 0.001). The mean decrease in perfusion is 50.79% immediately after treatment and is 32.14% at 4 weeks post-treatment. Immediately after treatment, the perfusion rate decreased rapidly. Following this, there was a partial recovery in perfusion rate up to 3 days post-treatment, followed by a plateau in the perfusion from 3 days to 4 weeks. CONCLUSIONS: This study demonstrated that a single PUT treatment could significantly reduce blood perfusion by 32.14% in the skin for up to 4 weeks. With unique advantages such as low laser fluence as compared with photothermolysis and agent-free treatment as compared with photodynamic therapy, PUT holds the potential to be developed into a new tool for the treatment of cutaneous vascular lesions. Lasers Surg. Med. © 2020 Wiley Periodicals, LLC.

CD26 Identifies a Subpopulation of Fibroblasts that Produce the Majority of Collagen during Wound Healing in Human Skin.

Fibroblasts produce collagens and other proteins that form the bulk of the extracellular matrix (ECM) in connective tissues. Emerging data point to functional heterogeneity of fibroblasts. However, the lack of subtype-specific markers hinders our understanding of the different roles of fibroblasts in ECM biology, wound healing, diseases, and aging. We have investigated the utility of the cell surface protein CD26 to identify functionally distinct fibroblast subpopulations in human skin. Using flow cytometry and immunohistology, we found that CD26, in combination with the cell surface glycoprotein CD90, identifies a distinct subpopulation of cells, which express relatively high levels of COL1A1, a hallmark of fibroblasts. Importantly, the population of CD26+ fibroblasts is selectively increased after wounding of human skin. These cells account for the majority of COL1A1 expression during the ECM remodeling phase of healing. The proportion of CD26+ fibroblasts in the skin of young and aged individuals is similar, indicating that the loss of collagen production during aging does not involve selective reduction of CD26+ fibroblasts. In culture, the majority of freshly isolated CD26- fibroblasts gain expression of CD26+. Taken together, these data provide a foundation for targeting CD26+ fibroblasts to modulate wound healing in human skin.

Reduced cell cohesiveness of outgrowths from eccrine sweat glands delays wound closure in elderly skin.

Human skin heals more slowly in aged vs. young adults, but the mechanism for this delay is unclear. In humans, eccrine sweat glands (ESGs) and hair follicles underlying wounds generate cohesive keratinocyte outgrowths that expand to form the new epidermis. Here, we compared the re-epithelialization of partial-thickness wounds created on the forearm of healthy young (< 40 yo) and aged (> 70 yo) adults. Our results confirm that the outgrowth of cells from ESGs is a major feature of repair in young skin. Strikingly, in aged skin, although ESG density is unaltered, less than 50% of the ESGs generate epithelial outgrowths during repair (vs. 100% in young). Surprisingly, aging does not alter the wound-induced proliferation response in hair follicles or ESGs. Instead, there is an overall reduced cohesiveness of keratinocytes in aged skin. Reduced cell-cell cohesiveness was most obvious in ESG-derived outgrowths that, when present, were surrounded by unconnected cells in the scab overlaying aged wounds. Reduced cell-cell contact persisted during the repair process, with increased intercellular spacing and reduced number of desmosomes. Together, reduced outgrowths of ESG (i) reduce the initial number of cells participating in epidermal repair, (ii) delay wound closure, and (iii) lead to a thinner repaired epidermis in aged vs. young skin. Failure to form cohesive ESG outgrowths may reflect impaired interactions of keratinocytes with the damaged ECM in aged skin. Our findings provide a framework to better understand the mediators of delayed re-epithelialization in aging and further support the importance of ESGs for the repair of human wounds.

A gender-based comparison of pain tolerance during pulsed dye laser therapy.

BACKGROUND: Cosmetic laser treatments are frequently performed without anesthesia in the clinic setting and there is a need to better understand the factors that may impact patient pain levels during these procedures. There has been prior research suggesting that there are significant gender-based differences in pain experiences with a variety of interventions. AIMS: We sought to examine the influence of gender and specific emotional factors on pain perception during pulsed dye laser treatments. PATIENTS/METHODS: We conducted a questionnaire-based study of 84 adult patients (42 males and 42 females) who underwent facial pulsed dye laser treatments in our clinic for cosmetic purposes. Questionnaires were completed by each patient after his or her initial laser treatment and patients were queried as to their perceived levels of pain during the procedure. Additional information regarding quality of life measures and patient motivation was also collected. RESULTS: Contrary to prior research suggesting lower pain thresholds for women in other clinical or experimental settings, we found no statistically significant differences in mean pain levels reported between patients of each gender. There was a trend toward females being somewhat more likely than males to see the pain of the treatment as justified for an improvement in appearance. CONCLUSIONS: Patient motivation and pain tolerance levels may be similar between genders among patients undergoing non-invasive cosmetic procedures. Clinicians may, therefore, expect patients of either gender to tolerate such treatments equally well.

Enhancing structural support of the dermal microenvironment activates fibroblasts, endothelial cells, and keratinocytes in aged human skin in vivo.

The dermal extracellular matrix (ECM) provides strength and resiliency to skin. The ECM consists mostly of type I collagen fibrils, which are produced by fibroblasts. Binding of fibroblasts to collagen fibrils generates mechanical forces, which regulate cellular morphology and function. With aging, collagen fragmentation reduces fibroblast-ECM binding and mechanical forces, resulting in fibroblast shrinkage and reduced function, including collagen production. Here, we report that these age-related alterations are largely reversed by enhancing the structural support of the ECM. Injection of dermal filler, cross-linked hyaluronic acid, into the skin of individuals over 70 years of age stimulates fibroblasts to produce type I collagen. This stimulation is associated with localized increase in mechanical forces, indicated by fibroblast elongation/spreading, and mediated by upregulation of type II TGF-ß receptor and connective tissue growth factor. Interestingly, enhanced mechanical support of the ECM also stimulates fibroblast proliferation, expands vasculature, and increases epidermal thickness. Consistent with our observations in human skin, injection of filler into dermal equivalent cultures causes elongation of fibroblasts, coupled with type I collagen synthesis, which is dependent on the TGF-ß signaling pathway. Thus, fibroblasts in aged human skin retain their capacity for functional activation, which is restored by enhancing structural support of the ECM.

Eccrine sweat glands are major contributors to reepithelialization of human wounds.

Eccrine sweat glands are skin-associated epithelial structures (appendages) that are unique to some primates including humans and are absent in the skin of most laboratory animals including rodents, rabbits, and pigs. On the basis of the known importance of other skin appendages (hair follicles, apocrine glands, and sebaceous glands) for wound repair in model animals, the present study was designed to assess the role of eccrine glands in the repair of wounded human skin. Partial-thickness wounds were generated on healthy human forearms, and epidermal repair was studied in skin biopsy samples obtained at precise times during the first week after wounding. Wound reepithelialization was assessed using immunohistochemistry and computer-assisted 3-dimensional reconstruction of in vivo wounded skin samples. Our data demonstrate a key role for eccrine sweat glands in reconstituting the epidermis after wounding in humans. More specifically, (i) eccrine sweat glands generate keratinocyte outgrowths that ultimately form new epidermis; (ii) eccrine sweat glands are the most abundant appendages in human skin, outnumbering hair follicles by a factor close to 3; and (iii) the rate of expansion of keratinocyte outgrowths from eccrine sweat glands parallels the rate of reepithelialization. This novel appreciation of the unique importance of eccrine sweat glands for epidermal repair may be exploited to improve our approaches to understanding and treating human wounds.

Direct quantitative comparison of molecular responses in photodamaged human skin to fractionated and fully ablative carbon dioxide laser resurfacing.

BACKGROUND: Fractionated ablative laser resurfacing has become a widely used treatment modality. Its clinical results are often found to approach those of traditional fully ablative laser resurfacing. OBJECTIVE: To directly compare the molecular changes that result from fractionated and fully ablative carbon dioxide (CO(2)) laser resurfacing in photodamaged human skin. METHODS AND MATERIALS: Photodamaged skin of 34 adult volunteers was focally treated at distinct sites with a fully ablative CO(2) laser and a fractionated CO(2) laser. Serial skin samples were obtained at baseline and several time points after treatment. Real-time reverse transcriptase polymerase chain reaction technology and immunohistochemistry were used to quantify molecular responses to each type of laser treatment. RESULTS: Fully ablative and fractionated CO(2) laser resurfacing induced significant dermal remodeling and collagen induction. After a single treatment, fractionated ablative laser resurfacing resulted in collagen induction that was approximately 40% to 50% as pronounced as that induced by fully ablative laser resurfacing. CONCLUSIONS: The fundamental cutaneous responses that result from fully ablative and fractionated carbon dioxide laser resurfacing are similar but differ in magnitude and duration, with the fully ablative procedure inducing relatively greater changes including more pronounced collagen induction. However, the molecular data reported here provide substantial support for fractionated ablative resurfacing as an effective treatment modality for improving skin texture.

Photothermolysis of sebaceous glands in human skin ex vivo with a 1,708 nm Raman fiber laser and contact cooling.

BACKGROUND AND OBJECTIVES: Wavelengths near ∼1,720 nm are of interest for targeting fat/lipid-rich tissues due to the high absorption coefficient of human fat and low water scattering and absorption. In this study, a 1,708 nm laser was built and shown to selectively target fat/lipid adjacent to porcine heart and dermis and then used to damage dermal sebaceous glands in human skin. STUDY DESIGN AND MATERIALS: An all-fiber 1,708 nm laser with ∼4 W maximum power was designed and built. Selectivity for targeting fat/lipid was studied by exposing porcine heart and skin tissue cross-sections to the 1,708 nm laser. Human skin treatments to damage sebaceous glands were performed both with and without cold window cooling. Histochemical evaluation on the frozen sections was performed using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. RESULTS: Histochemical analysis of porcine tissue cross-sections showed that 1,708 nm laser can selectively damage pericardial fat(heart) and subcutaneous fat(skin) with little to no damage to the myocardium and the dermis, respectively. In human skin, histochemical evaluation without contact cooling showed damage to both epidermis and dermis. With cooling, epidermis was spared and damage was observed in dermis extending ∼0.4-1.65 mm from the skin surface at an average laser fluence of ∼80 J/cm(2). Selective damage of sebaceous glands was suggested but not definitively demonstrated. CONCLUSIONS: We have developed an all-fiber 1,708 nm laser capable of damaging majority of the sebaceous glands in the dermis and thus may have potential applications in the treatment of conditions such as acne vulgaris whose pathophysiology involves disorders of sebaceous glands.

Spatial-temporal modulation of CCN proteins during wound healing in human skin in vivo.

CCN proteins are important modulators of development and function of adult organs. In this study, we examined the localization and expression of the six CCN family members in normal adult human skin and during wound healing in vivo. Transcript and protein expression were studied by laser-capture microdissection-coupled real-time PCR and immunohistochemistry, respectively. Our results demonstrate that CCN1, CCN4, and CCN6 are expressed at relatively low levels in normal human skin. CCN2, CCN3, and CCN5 are the most highly expressed transcripts in the epidermis. CCN3 and CCN5 proteins are prominent in epidermal keratinocytes, whereas CCN2 is primarily expressed in melanocytes. Differential expression within epidermal layers suggests that CCN3 and CCN5 are linked with keratinocyte differentiation. CCN2, CCN3 and CCN5, are the three most highly expressed transcripts in the dermis. Their respective proteins are produced to various extents by dermal fibroblasts, blood vessels, eccrine sweat glands and hair follicles. We find that most CCN family members are temporally and specifically regulated during different phases (inflammation, proliferation, and remodeling) of partial thickness wound repair. By highlighting spatial-temporal regulations of CCN family member expression in relation to cell proliferation and differentiation, our results suggest a diverse range of functions for CCN proteins in both epidermal and dermal cells, and provides a solid reference for interpretation of future studies aimed at understanding the role of CCN proteins in human skin physiology and diseases.