Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver.

Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

The Yucatan miniature swine as a model for post-inflammatory hyperpigmentation.

Post-inflammatory hyperpigmentation (PIH) is a hypermelanosis that often occurs secondary to skin irritation or injury, especially in darker skin tones, for which there is currently a lack of effective treatment options. Few preclinical models are available to study PIH. Here, we show that the Yucatan miniature pig consistently develops PIH after skin injuries. Skin wounds were produced on Yucatan pigs by needle punches, full-thickness excisions, or burns. Wound sites were monitored and photographed regularly. Tissue samples were collected after 24 weeks and processed for histology/immunohistochemistry. Skin pigmentation and histologic changes were quantified by computer-assisted image analyses. All injury methods resulted in hyperpigmentation. Melanin content at the histologic level was quantified in the larger (burn and excision) wounds, showing a significant increase compared to uninjured skin. Increased melanin was found for both epidermal and dermal regions. Dermal melanin deposits were primarily clustered around the papillary vasculature, and were associated not with melanocytes but with leukocytes. The Yucatan miniature pig model recapitulates key clinical and histologic features of PIH in humans, including skin hyperpigmentation at both gross and histologic levels, and persistence of dermal melanin subsequent to injury. This model could be used to further our understanding of the etiology of PIH, and for new therapy development.

Plantar Skin Exhibits Altered Physiology, Constitutive Activation of Wound-Associated Phenotypes, and Inherently Delayed Healing.

Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. The skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from nonplantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating nonhealing wounds.

An antimicrobial blue light device to manage infection at the skin-implant interface of percutaneous osseointegrated implants.

Antimicrobial blue light (aBL) is an attractive option for managing biofilm burden at the skin-implant interface of percutaneous osseointegrated (OI) implants. However, marketed aBL devices have both structural and optical limitations that prevent them from being used in an OI implant environment. They must be handheld, preventing even irradiation of the entire skin-implant interface, and the devices do not offer sufficient optical power outputs required to kill biofilms. We present the developmental process of a unique aBL device that overcomes these limitations. Four prototypes are detailed, each being a progressive improvement from the previous iteration as we move from proof-of-concept to in vivo application. Design features focused on a cooling system, LED orientation, modularity, and "sheep-proofing". The final prototype was tested in an in vivo OI implant sheep model, demonstrating that it was structurally and optically adequate to address biofilm burdens at the skin-implant of percutaneous OI implants. The device made it possible to test aBL in the unique OI implant environment and compare its efficacy to clinical antibiotics-data which had not before been achievable. It has provided insight into whether or not continued pursual of light therapy research for OI implants, and other percutaneous devices, is worthwhile. However, the device has drawbacks concerning the cooling system, complexity, and size if it is to be translated to human clinical trials. Overall, we successfully developed a device to test aBL therapy for patients with OI implants and helped progress understanding in the field of infection management strategies.

Comparison of Staphylococcus aureus tolerance between antimicrobial blue light, levofloxacin, and rifampin.

Background: Bacterial biofilms readily develop on all medical implants, including percutaneous osseointegrated (OI) implants. With the growing rate of antibiotic resistance, exploring alternative options for managing biofilm-related infections is necessary. Antimicrobial blue light (aBL) is a unique therapy that can potentially manage biofilm-related infections at the skin-implant interface of OI implants. Antibiotics are known to have antimicrobial efficacy disparities between the planktonic and biofilm bacterial phenotypes, but it is unknown if this characteristic also pertains to aBL. In response, we developed experiments to explore this aspect of aBL therapy. Methods: We determined minimum bactericidal concentrations (MBCs) and antibiofilm efficacies for aBL, levofloxacin, and rifampin against Staphylococcus aureus ATCC 6538 planktonic and biofilm bacteria. Using student t-tests (p < 0.05), we compared the efficacy profiles between the planktonic and biofilm states for the three independent treatments and a levofloxacin + rifampin combination. Additionally, we compared antimicrobial efficacy patterns for levofloxacin and aBL against biofilms as dosages increased. Results: aBL had the most significant efficacy disparity between the planktonic and biofilm phenotypes (a 2.5 log10 unit difference). However, further testing against biofilms revealed that aBL had a positive correlation between increasing efficacy and exposure time, while levofloxacin encountered a plateau. While aBL efficacy was affected the most by the biofilm phenotype, its antimicrobial efficacy did not reach a maximum. Discussion/conclusion: We determined that phenotype is an important characteristic to consider when determining aBL parameters for treating OI implant infections. Future research would benefit from expanding these findings against clinical S. aureus isolates and other bacterial strains, as well as the safety of long aBL exposures on human cells.

The case for considering volar skin in a "separate status" for wound healing.

Foot ulcers, particularly in the diabetic setting, are a major medical and socioeconomic challenge. While the effects of diabetes and its various sequelae have been extensively studied, in the wound field it is commonly assumed that the wound healing process is essentially identical between different skin types, despite the many well-known specializations in palmoplantar skin, most of which are presumed to be evolutionary adaptations for weightbearing. This article will examine how these specializations could alter the wound healing trajectory and contribute to the pathology of foot ulcers.

Antimicrobial blue light as a biofilm management therapy at the skin-implant interface in an ex vivo percutaneous osseointegrated implant model.

Biofilm contamination is often present at the skin-implant interface of transfemoral osseointegrated implants leading to frequent infection, irritation, and discomfort. New biofilm management regimens are needed as the current standard of washing the site with soap and water is inadequate to manage infection rates. We investigated the potential of antimicrobial blue light, which has reduced risk of resistance development and broad antimicrobial mechanisms. Our lab developed an antimicrobial blue light (aBL) device uniquely designed for an ex vivo system based on an established ovine osseointegrated (OI) implant model with Staphylococcus aureus ATCC 6538 biofilms as initial inocula. Samples were irradiated with aBL or washed for three consecutive days after which they were quantified. Colony-forming unit (CFU) counts were compared with a control group (bacterial inocula without treatment). After 1 day, aBL administered as a single 6 h dose or two 1 h doses spaced 6 h apart both reduced the CFU count by 1.63 log10 ± 0.02 CFU. Over 3 days of treatment, a positive aBL trend was observed with a maximum reduction of ~2.7 log10 CFU following 6 h of treatment, indicating a relation between multiple days of irradiation and greater CFU reductions. aBL was more effective at reducing the biofilm burden at the skin-implant interface compared with the wash group, demonstrating the potential of aBL as a biofilm management option.

Boot camp: Training and dressing regimens for modeling plantar wounds in the swine.

Foot ulceration annually affects millions of patients and accounts for billions of dollars in medical expenses in the US alone. Many previous studies have investigated co-morbidities associated with impaired healing, such as microbial infection, compromised circulation, and diabetes. By comparison, little is known about how wound healing proceeds in plantar skin, despite its many unique specializations related to its load-bearing function. One of the main challenges in modeling plantar wounds is the difficulty in maintaining wound dressings, as animals generally have a low tolerance to wearing bandages on their feet. With assistance from the MGH Center for Comparative Medicine, we developed a positive reinforcement-based behavioral training regimen that successfully induced tolerance for plantar dressings in swine, which is a critical first step towards enabling in vivo study of the wound healing process in this highly specialized skin area. This training program will be described in detail in this manuscript.

Research protocol to evaluate the effectiveness of shockwave therapy, photobiomodulation and physical therapy in the management of non-insertional Achilles tendinopathy in runners: a randomised control trial with elective cross-over design.

Background: Achilles tendinopathy (AT) is a common overuse injury in runners. While the mainstay of treatment for AT is tendon loading exercises (physical therapy and exercise programme (EXER)), some patients have refractory symptoms. Extracorporeal shockwave therapy (ESWT) and photobiomodulation therapy (PBMT) have each been evaluated to facilitate tendon healing; the influence of combining treatments is unknown and limited studies have been completed in runners. This randomised control study, with an elective cross-over at 3 months, will evaluate the efficacy of three forms of treatment of non-insertional AT: (1) EXER (loading programme specific to Achilles tendon combined with physical therapy); (2) EXER and ESWT; (3) EXER, ESWT and PBMT. Sixty runners will be assigned using block randomisation into one of three treatment groups (n=20). After 3 months, each participant may elect a different treatment than previously assigned and will be followed for an additional 3 months. The EXER Achilles loading programme will be standardised using the Silbernagel at-home programme. The primary outcome of interest is treatment group responses using the Victorian Institute of Sports Assessment-Achilles (VISA-A) Score. Secondary outcomes include the Patient-Reported Outcomes Measurement Information System-29 questions, the University of Wisconsin Running Injury and Recovery Index, heel raise to fatigue test, hopping test and ultrasound measurements. We will also capture patient preference and satisfaction with treatment. We hypothesise that the cohorts assigned EXER+ESWT+PBMT and EXER+ESWT will see greater improvements in VISA-A than the EXER cohort, and the largest gains are anticipated in combining ESWT+PBMT. The elective cross-over phase will be an exploratory study and will inform us whether patient preference for treatment will impact the treatment response. Trial registration number: NCT04725513.

Local vasoregulative interventions impact drug concentrations in the skin after topical laser-assisted delivery.

INTRODUCTION: The ability of ablative fractional lasers (AFL) to enhance topical drug uptake is well established. After AFL delivery, however, drug clearance by local vasculature is poorly understood. Modifications in vascular clearance may enhance AFL-assisted drug concentrations and prolong drug dwell time in the skin. Aiming to assess the role and modifiability of vascular clearance after AFL-assisted delivery, this study examined the impact of vasoregulative interventions on AFL-assisted 5-fluorouracil (5-FU) concentrations in in vivo skin. METHODS: 5-FU uptake was assessed in intact and AFL-exposed skin in a live pig model. After fractional CO2 laser exposure (15 mJ/microbeam, 5% density), vasoregulative intervention using topical brimonidine cream, epinephrine solution, or pulsed dye laser (PDL) was performed in designated treatment areas, followed by a single 5% 5-FU cream application. At 0, 1, 4, 48, and 72 h, 5-FU concentrations were measured in 500 and 1500 µm skin layers by mass spectrometry (n = 6). A supplemental assessment of blood flow following AFL ± vasoregulation was performed using optical coherence tomography (OCT) in a human volunteer. RESULTS: Compared to intact skin, AFL facilitated a prompt peak in 5-FU delivery that remained elevated up to 4 hours (1500 µm: 1.5 vs. 31.8 ng/ml [1 hour, p = 0.002]; 5.3 vs. 14.5 ng/ml [4 hours, p = 0.039]). However, AFL's impact was transient, with 5-FU concentrations comparable to intact skin at later time points. Overall, vasoregulative intervention with brimonidine or PDL led to significantly higher peak 5-FU concentrations, prolonging the drug's dwell time in the skin versus AFL delivery alone. As such, brimonidine and PDL led to twofold higher 5-FU concentrations than AFL alone in both skin layers by 1 hour (e.g., 500 µm: 107 ng/ml [brimonidine]; 96.9 ng/ml [PDL], 46.6 ng/ml [AFL alone], p ≤ 0.024), and remained significantly elevated at 4 hours (p ≤ 0.024). A similar pattern was observed for epinephrine, although trends remained nonsignificant (p ≥ 0.09). Prolonged 5-FU delivery was provided by PDL, resulting in sustained drug deposition compared to AFL alone at both 48 and 72 hours in the superficial skin layer (p ≤ 0.024). Supporting drug delivery findings, OCT revealed that increases in local blood flow after AFL were mitigated in test areas also exposed to PDL, brimonidine, or epinephrine, with PDL providing the greatest, sustained reduction in flow over 48 hours. CONCLUSION: Vasoregulative intervention in conjunction with AFL-assisted delivery enhances and prolongs 5-FU deposition in in vivo skin.

Laser Immunotherapy: A Potential Treatment Modality for Keratinocyte Carcinoma.

The role of the immune system in cancer growth is well recognized and the development of immunotherapy represents a breakthrough in cancer treatment. Recently, the use of systemic immunotherapy was extended to keratinocyte carcinoma (KC), specifically locally advanced and metastasizing basal and squamous cell carcinoma. However, since most KC lesions are non-aggressive, systemic treatment with associated side effects is rarely justified. Conversely, topical immunotherapy with imiquimod remains restricted to premalignant and superficial lesions. Use of laser in the treatment of KC has evolved from physical tumor destruction and laser-assisted drug delivery to laser-mediated immune modulation. Evidence indicates that laser monotherapy can lead to immune cell infiltration, tumor reduction and resistance to tumor re-inoculation. Combining laser with immunotherapeutic agents, termed laser immunotherapy (LIT), may further potentiate immune activation and tumor response. Studies on LIT show not only direct anti-tumor effects but systemic adaptive immunity, illustrated by the prevention of tumor recurrence and regression in distant untreated tumors. These findings imply a therapeutic potential for both local and metastatic disease. This work provides rationales for immune-based treatment of KC and presents the current status of KC immunotherapy. Aiming to expand the field of KC immunotherapy, the review discusses the literature on immune activation following laser monotherapy and LIT.

MagneTEskin-Reconstructing skin by magnetically induced assembly of autologous microtissue cores.

Skin wounds are immense medical and socioeconomic burdens, and autologous skin grafting remains the gold standard for wound repair. We recently found that full-thickness micro skin tissue columns (MSTCs) can be harvested with minimal donor site morbidity, and that MSTCs applied to wounds "randomly" (without maintaining their natural epidermal-dermal orientation) can accelerate re-epithelialization. However, despite MSTCs containing all the cellular and extracellular contents of full-thickness skin, normal dermal architecture was not restored by random MSTCs. In this study, we developed a magnetically induced assembly method to produce constructs of densely packed, oriented MSTCs that closely resemble the overall architecture of full-thickness skin to test the hypothesis that maintaining MSTCs' orientation could further hasten healing and restore a normal dermis. Our method led to faster and more orderly re-epithelialization but unexpectedly did not improve the retention of dermal architecture, which reveals a hitherto unappreciated role for tissue morphology in determining dermal remodeling outcomes.

Photobiomodulation Response From 660 nm is Different and More Durable Than That From 980 nm.

BACKGROUND AND OBJECTIVES: Photobiomodulation (PBM) therapy uses light at various wavelengths to stimulate wound healing, grow hair, relieve pain, and more-but there is no consensus about optimal wavelengths or dosimetry. PBM therapy works through putative, wavelength-dependent mechanisms including direct stimulation of mitochondrial respiration, and/or activation of transmembrane signaling channels by changes in water activity. A common wavelength used in the visible red spectrum is ~660 nm, whereas recently ~980 nm is being explored and both have been proposed to work via different mechanisms. We aimed to gain more insight into identifying treatment parameters and the putative mechanisms involved. STUDY DESIGN/MATERIALS AND METHODS: Fluence-response curves were measured in cultured keratinocytes and fibroblasts exposed to 660 or 980 nm from LED sources. Metabolic activity was assessed using the MTT assay for reductases. ATP production, a major event triggered by PBM therapy, was assessed using a luminescence assay. To measure the role of mitochondria, we used an ELISA to measure COX-1 and SDH-A protein levels. The respective contributions of cytochrome c oxidase and ATP synthase to the PBM effects were gauged using specific inhibitors. RESULTS: Keratinocytes and fibroblasts responded differently to exposures at 660 nm (red) and 980 nm (NIR). Although 980 nm required much lower fluence for cell stimulation, the resulting increase in ATP levels was short-term, whereas 660 nm stimulation elevated ATP levels for at least 24 hours. COX-1 protein levels were increased following 660 nm treatment but were unaffected by 980 nm. In fibroblasts, SDH-A levels were affected by both wavelengths, whereas in keratinocytes only 660 nm light impacted SDH-A levels. Inhibition of ATP synthase nearly completely abolished the effects of both wavelengths on ATP synthesis. Interestingly, inhibiting cytochrome c oxidase did not prevent the rise in ATP levels in response to PBM treatment. CONCLUSION: To the best of our knowledge, this is the first demonstration of differing kinetics in response to PBM therapy at red versus NIR wavelength. We also found cell-type-specific differences in PBM therapy response to the two wavelengths studied. These findings confirm that different response pathways are involved after 660 and 980 nm exposures and suggest that 660 nm causes a more durable response. © 2021 Wiley Periodicals LLC.

Multi-faceted enhancement of full-thickness skin wound healing by treatment with autologous micro skin tissue columns.

Impaired wound healing is an immense medical challenge, and while autologous skin grafting remains the "gold-standard" therapeutic option for repairing wounds that cannot be closed by primary or secondary intention, it is limited by substantial donor site morbidity. We previously developed the alternative approach of harvesting full-thickness skin tissue in the form of "micro skin tissue columns" (MSTCs), without causing scarring or any other long-term morbidity. In this study we investigated how MSTC treatment affects the different cellular processes involved in wound healing. We found that MSTC-derived cells were able to remodel and repopulate the wound volume, and positively impact multiple aspects of the wound healing process, including accelerating re-epithelialization by providing multiple cell sources throughout the wound area, increasing collagen deposition, enhancing dermal remodeling, and attenuating the inflammatory response. These effects combined to enhance both epidermal and dermal wound healing. This MSTC treatment approach was designed for practical clinical use, could convey many benefits of autologous skin grafting, and avoids the major drawback of donor site morbidity.

Construction and validation of UV-C decontamination cabinets for filtering facepiece respirators.

We present evidence-based design principles for three different UV-C based decontamination systems for N95 filtering facepiece respirators (FFRs) within the context of the SARS-CoV-2 outbreak of 2019-2020. The approaches used here were created with consideration for the needs of low- and middle-income countries (LMICs) and other under-resourced facilities. As such, a particular emphasis is placed on providing cost-effective solutions that can be implemented in short order using generally available components and subsystems. We discuss three optical designs for decontamination chambers, describe experiments verifying design parameters, validate the efficacy of the decontamination for two commonly used N95 FFRs (3M, #1860 and Gerson #1730), and run mechanical and filtration tests that support FFR reuse for at least five decontamination cycles.

Injectable slurry for selective destruction of neck adipose tissue in New Zealand obese mouse model.

PURPOSE: Increased neck circumference is a major risk factor for obstructive sleep apnea (OSA). New data suggest that increased adipose tissue in the neck may be a contributory cause of OSA. The aim of this study was to investigate safety and efficacy of a recently developed injectable ice slurry in selective reduction of neck adipose tissue in a mouse model. METHODS: We used the New Zealand obese mice that have increased volume of anterior neck fat, and are commonly used in OSA studies. MRI imaging was used to measure changes in fat tissue volume. RESULTS: Thirty animals were used in this study. Volumetric measurements in MRI images showed thatchanges in anterior neck adipose tissue volume from baseline in treated mice was significantly different in comparison with the control group (-1.09/kg ± 0.33/kg vs 0.68/kg ± 0.37/kg; p < 0.01 by two-tailed Student's t test). Histological analysis of samples from the treated area of the neck did not show scarring or damage to the surrounding tissues. CONCLUSIONS: Injection of ice slurry safely, effectively, and selectively reduces upper airway fat in New Zealand obese mice without scarring or damage to surrounding tissue. Our results suggest that slurry injection may be a novel and minimally invasive method of removing neck adipose tissue. This intervention should be further investigated to determine its suitability for treatment of OSA.

Skin Microcolumns as a Source of Paracrine Signaling Factors.

Objective: We recently developed the approach of using "microcolumns" of autologous full-thickness skin tissue for wound repair. The small size of these micro skin tissue columns (MSTCs, ∼0.5 mm in diameter) allows donor sites to heal quickly without scarring. Treatment with MSTCs significantly accelerate wound healing, and suppled various skin cell types and skin structures to replenish the wound volume. This technology is now starting clinical use. In this study, we investigate whether MSTCs may also influence wound healing by releasing soluble signaling factors. Approach: Freshly harvested MSTCs were incubated in culture medium for 24 h. The conditioned medium was collected and tested for its effects on migration and proliferation of human dermal fibroblasts, and its ability to induce tube formation by human umbilical vein endothelial cells (HUVECs). Proteins released into the conditioned medium were characterized by multiplex enzyme-linked immunosorbent assay (ELISA), and compared with medium conditioned by an equivalent mass of intact full-thickness skin. Results: MSTC-conditioned medium increased fibroblast migration and proliferation, as well as HUVEC tube formation. MSTCs released many soluble factors known to play prominent roles in wound healing. A subset of proteins showed significantly different release profiles compared with intact full-thickness skin. Innovation: The technology for harvesting and using MSTCs to augment wound healing was recently developed as an alternative to conventional autologous skin grafting. This study shows that MSTCs could also function as "cytokine factories." Conclusion: In addition to supplying autologous cells to repopulate the wound volume, MSTCs can also function as a source of growth factors and cytokines to further enhance wound healing.

When Wounds Are Good for You: The Regenerative Capacity of Fractional Resurfacing and Potential Utility in Chronic Wound Prevention.

Significance: Fractional resurfacing involves producing arrays of microinjuries on the skin, by thermal or mechanical means, to trigger tissue regeneration. Originally developed for cosmetic enhancement, fractional resurfacing induces a broad array of improvements in the structural and functional qualities of the treated skin and is especially effective at returning defective skin to a more normal state. In addition to fascinating questions about the nature of this remarkable regenerative capacity, there may be potential utility in ulcer prevention by halting or even reversing the progressive decline in overall skin quality that usually precedes chronic wound development. Recent Advances: Photoaging and scarring are the two skin defects most commonly treated by fractional resurfacing, and the treatment produces profound and long-lasting improvements in skin quality, both clinically and at the cellular/histologic level. Chronic wounds usually occur in skin that is compromised by various pathologic factors, and many of the defects found in this ulcer-prone skin are similar to those that have seen improvements after fractional resurfacing. Critical Issues: The mechanisms responsible for the regenerative capacity of fractional resurfacing are mostly unknown, as is how ulcer-prone skin, which is usually afflicted by stressors external to the skin tissue itself, would respond to fractional resurfacing. Future Directions: Better understanding of the cellular and molecular mechanisms underlying the unique healing response to fractional resurfacing could reveal fundamental information about adult tissue regeneration, lead to improvements in current applications, as well as new therapies in other pathologic conditions.

Apparatus for Harvesting Tissue Microcolumns.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Laser-assisted delivery of synergistic combination chemotherapy in in vivo skin.

The effectiveness of topical drugs for treatment of non-melanoma skin cancer is greatly reduced by insufficient penetration to deep skin layers. Ablative fractional lasers (AFLs) are known to enhance topical drug uptake by generating narrow microchannels through the skin, but information on AFL-drug delivery in in vivo conditions is limited. In this study, we examined pharmacokinetics, biodistribution and toxicity of two synergistic chemotherapy agents, cisplatin and 5-fluorouracil (5-FU), following AFL-assisted delivery alone or in combination in in vivo porcine skin. Detected at 0-120 h using mass spectrometry techniques, we demonstrated that fractional CO2 laser pretreatment (196 microchannels/cm2, 852 µm ablation depth) leads to rapid drug uptake in 1500 µm deep skin layers, with a sixfold enhancement in peak cisplatin concentrations versus non-laser-treated controls (5 h, P = 0.005). Similarly, maximum 5-FU deposition was measured within an hour of AFL-delivery, and exceeded peak deposition in non-laser-exposed skin that had undergone topical drug exposure for 5 days. Overall, this accelerated and deeper cutaneous drug uptake resulted in significantly increased inflammatory and histopathological effects. Based on clinical scores and transepidermal water loss measurement, AFL intensified local toxic responses to drugs delivered alone and in combination, while systemic drug exposure remained undetectable. Quantitative histopathologic analyses correspondingly revealed significantly reduced epidermal proliferation and greater cellular apoptosis after AFL-drug delivery; particularly after combined cisplatin + 5-FU exposure. In sum, by overcoming the primary limitation of topical drug penetration and providing accelerated, enhanced and deeper delivery, AFL-assisted combination chemotherapy may represent a promising treatment strategy for non-melanoma skin cancer.