Inhibition of cyclooxygenase‐1 and ‐2 activity in keratinocytes inhibits PGE2 formation and impairs vascular endothelial growth factor release and neovascularisation in skin wounds

Inhibition of cyclooxygenase (Cox) enzymatic activity by non‐steroidal anti‐inflammatory drugs (NSAIDs) provides the molecular basis of analgesia following wounding or surgery. This study investigated the role of Cox activity in the regulation of vascular endothelial growth factor (VEGF) expression in keratinocytes and the formation of new blood vessels in acute wounds in mice. To this end, human HaCaT keratinocytes were stimulated with epidermal growth factor (EGF). EGF increased Cox‐1 mRNA in the presence of the constitutively expressed Cox‐1 protein in keratinocytes. EGF coinduced Cox‐2 and VEGF₁₆₅ mRNA and protein expression and an accumulation of prostaglandin E₂ (PGE₂) in cell culture supernatants. Inhibition of Cox isozyme activity by Cox‐1 and ‐2 siRNA or ibuprofen reduced PGE₂ and VEGF₁₆₅ release from keratinocytes. In a mouse model of excisional wound healing, Cox‐2 and VEGF₁₆₅ expression were colocalized in the granulation tissue of acute wounds. Oral treatment of mice with the Cox‐1 and ‐2 inhibitor diclofenac was associated with reduced levels of VEGF₁₆₅ protein and an impaired blood vessel formation in acute wound tissue. In summary, our data suggest that a reduction of PGE₂‐triggered VEGF₁₆₅ protein expression in wound keratinocytes is likely to contribute to the observed impairment of wound neovascularisation upon Cox inhibition.     

Biomimetic bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid biopolymer as a scaffold for skin equivalent tissue engineering

In order to develop an adequate scaffold for skin tissue engineering, a bilayered gelatin-chondroitin 6 sulfate-hyaluronic acid membrane with a different pore size on either side was prepared. A rete ridges-like topographic microporous structure, which provided the paracrine crosstalk in the epithelial-mesenchymal interactions, was formed. Chondroitin-6-sulfate and hyaluronic acid were incorporated within the gelatin membrane to mimic skin composition and create an appropriate microenvironment for cell proliferation, differentiation, and migration. In the study, the lower layer of the membrane (pore size: 150 microm) was seeded with dermal fibroblasts and acted as the feeder layer for keratinocyte inoculation. Meanwhile, the upper layer (pore size: 20-50 microm) was seeded with keratinocytes for epidermalization. The dermal fibroblasts were dynamically seeded in a self-designed spinner flask for more even cell distribution. The keratinocytes were cultured in submerged conditions for 5 days and then in an air-liquid interface condition for further differentiation. After being cultured for 21 days, the upper layer, seeded with keratinocytes, developed into an epidermis-like structure while the lower part, which was seeded with dermal fibroblasts developed into a dermis-like structure. A histological examination and immunostain were used to prove that keratinocytes maintain their phenotype and stratified epidermis layers were formed within 21 days. In brief, the bilayered skin substitute with biological dermal analog and epidermal structure was successfully fabricated. From this study, we can suggest that the culture model is suitable for autologous skin equivalent preparation.     

Evaluation of Permacol as a cultured skin equivalent

Skin loss following severe burn requires prompt wound closure to avoid such complications as fluid and electrolyte imbalance, infection, immune suppression, and pain. In clinical situations in which insufficient donor skin is available, the development of cultured skin equivalents (dermal matrices seeded with keratinocytes and fibroblasts) may provide a useful alternative. The aim of this study was to assess the suitability of a porcine-derived dermal collagen matrix (Permacol™) to function as a cultured skin equivalent in supporting the growth of keratinocytes in vitro and providing cover to full thickness wounds in the BALB C/nude mouse model. A histological comparison was against Glycerol treated-Ethylene Oxide Sterilised Porcine Dermis (Gly-EO Dermis) which has successfully been used as a cultured skin equivalent in previous studies. Both Gly-EO Dermis and to a lesser extent Permacol™ were able to support the growth of cultured keratinocytes following a 16-day period of cell culture, however, this study was only able to demonstrate the presence of an epidermal layer on Gly-EO dermis 2 weeks after grafting onto full-thickness wounds in the BALB C/nude mouse model.     

Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin

BACKGROUND: Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. METHODS: Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. RESULTS: Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. CONCLUSIONS: Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.     

Differential effects of planktonic and biofilm MRSA on human fibroblasts

Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin‐resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell‐culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin‐resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin‐resistant S. aureus, namely limited migration followed by death. Enzyme‐linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm‐conditioned medium. Planktonic‐conditioned medium induced more interleukin‐6, interleukin‐8, vascular endothelial growth factor, transforming growth factor‐β1, heparin‐bound epidermal growth factor, matrix metalloproteinase‐1, and metalloproteinase‐3 production in fibroblasts than the biofilm‐conditioned medium. Biofilm‐conditioned medium induced more tumor necrosis factor‐α production in fibroblasts compared with planktonic‐conditioned medium, and suppressed metalloproteinase‐3 production compared with controls.     

Prostaglandin E2 and prostaglandin F2α differentially modulate matrix metabolism of human nucleus pulposus cells

Prostaglandin (PG) actions on disc metabolism are unclear even though certain PGs are highly expressed by disc cells under inflammatory conditions and nonsteroidal anti‐inflammatory drugs (NSAIDs) are frequently used to block PG production to treat back pain. Hence this study aimed to (1) quantify gene expression of arachidonic acid cascade components responsible for PG synthesis and (2) examine the effects of key PGs on disc matrix homeostasis. Microarray analysis revealed that inflammatory stress increases expression of synthases and receptors for prostaglandin E2 (PGE₂) and prostaglandin F2α (PGF_2α), resulting in elevated PGE₂ and PGF_2α production in conditioned media of disc cells. PGE₂ diminished disc cell proteoglycan synthesis, in a dose‐dependent manner. Semiquantitative RT‐PCR revealed differential effects of PGE₂ and PGF_2α on disc cell expression of key matrix structural genes, aggrecan, versican, collagens type I and II. PGE₂ and PGF_2α also decreased message for the anabolic factor, IGF‐1. PGE₂ decreased mRNA expression for the anti‐catabolic factor TIMP‐1 while PGF_2α increased mRNAs for catabolic factors MMP‐1 and MMP‐3. Thus, PGE₂ and PGF_2α may have an overall negative impact on disc matrix homeostasis, and the use of NSAIDs may impact disc metabolism as well as treat back pain. Published by Wiley Periodicals, Inc. J Orthop Res 28:1259–1266, 2010     

Increased activity of TRPV3 in keratinocytes in hypertrophic burn scars with postburn pruritus

Postburn pruritus is a common distressing sequela of burn wounds. Empirical antipruritic treatment often fails to have a satisfactory outcome, as the mechanism of it has not been fully elucidated. The aim of this study was to evaluate the manifestation of transient receptor potential vanilloid 3 (TRPV3), transient receptor potential ankyrin 1 (TRPA1), and other related receptors in postburn pruritus. Sixty‐five burn patients with (n = 40) or without (n = 25) pruritus were investigated, including skin biopsies. Keratinocytes and fibroblasts from skin biopsy samples were separated. Real time‐PCR showed that mRNA of TRPV3 was significantly increased in keratinocytes from pruritic burn scars than in keratinocytes from nonpruritic burn scars. With TRPV3 activation, intracellular Ca²⁺ concentrations were more significantly increased in keratinocytes from pruritic burn scars than in those from nonpruritic ones. Additionally, mRNA and protein levels of protease‐activated receptor 2 (PAR2) and neurokinin‐1 receptor (NK1R) were also significantly increased in pruritic burn scars. In conclusion, it was confirmed that TRPV3, PAR2, and NK1R were highly expressed in pruritic burn scars. These results may help determine a novel mechanism for postburn pruritus.     

071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

Deep dermal fibroblast profibrotic characteristics are enhanced by bone marrow–derived mesenchymal stem cells

Hypertrophic scars are a significant fibroproliferative disorder complicating deep injuries to the skin. We hypothesize that activated deep dermal fibroblasts are subject to regulation by bone marrow–derived mesenchymal stem cells (BM‐MSCs), which leads to the development of excessive fibrosis following deep dermal injury. We found that the expression of fibrotic factors was higher in deep burn wounds compared with superficial burn wounds collected from burn patients with varying depth of skin injury. We characterized deep and superficial dermal fibroblasts, which were cultured from the deep and superficial dermal layers of normal uninjured skin obtained from abdominoplasty patients, and examined the paracrine effects of BM‐MSCs on the fibrotic activities of the cells. In vitro, deep dermal fibroblasts were found higher in the messenger RNA (mRNA) levels of type 1 collagen, alpha smooth muscle actin, transforming growth factor beta, stromal cell–derived factor 1, and tissue inhibitor of metalloproteinase 1, an inhibitor of collagenase (matrix metalloproteinase 1). As well, deep dermal fibroblasts had low matrix metalloproteinase 1 mRNA, produced more collagen, and contracted collagen lattices significantly greater than superficial fibroblasts. By co‐culturing layered fibroblasts with BM‐MSCs in a transwell insert system, BM‐MSCs enhanced the fibrotic behavior of deep dermal fibroblasts, which suggests a possible involvement of BM‐MSCs in the pathogenesis of hypertrophic scarring.     

Novel model for evaluation of epidermal preservation and dermal collagen remodeling following photorejuvenation of human skin

BACKGROUND AND OBJECTIVES: In order to optimize photorejuvenation of human skin, a method must be developed to reliably compare the potential for epidermal preservation and dermal fibroblast stimulation of different laser devices and irradiation parameters. We describe a novel human skin tissue culture model developed for this purpose. MATERIALS AND METHODS: An artificial skin model, consisting of human keratinocytes in the epidermis and human fibroblasts and rat-tail collagen in the dermis, was cultured using the floating collagen gel (RAFT) method. Repetitive low-fluence Er:YAG laser irradiation was applied to test the applicability of our RAFT model for characterization of epidermal preservation and dermal fibroblast stimulation post-laser treatment. RESULTS: Histopathologic evaluation revealed a thin layer of epidermal keratinocyte preservation immediately after low fluence sub-ablative Er:YAG laser irradiation. One-week post-laser irradiation, the average increase in number of dermal fibroblasts as compared to control was statistically significant (P < 0.01). CONCLUSIONS: The RAFT model can be used to assess the potential for epidermal preservation and dermal fibroblast stimulation of different photorejuvenation devices and irradiation parameters and offers several advantages over traditional animal and human skin models.     

Tissue engineered artificial skin composed of dermis and epidermis

Abstract: We made an artificial skin comprised of a stratified layer of keratinocytes and a dermal matrix with a type I collagen containing fibroblasts. In this work, we showed keratinocyte behavior under primary culture, gel contractions varying with concentration of collagen solution, and cell growth plots in the collagen gel. The optimum behavior of dermal equivalent could be obtained using 3.0 mg/ml collagen solution and attached gel culture. The attached gel culture had a jumping effect of growth factor on cell growth at the lag phase. To develop the artificial skin, 1× 10⁵ cells/cm² of keratinocytes were cultured on the dermal equivalent at air-liquid interface. Finally, to overcome the problem that artificial skin of collagen gel was torn easily during suturing of grafting, we prepared histocompatible collagen mesh and attached the mesh to the bottom of the gel. Cultured artificial skins were successfully grafted onto rats.     

Response to tumor necrosis factor‐α mediated inflammation involving activation of prostaglandin E2 and Wnt signaling in nucleus pulposus cells

The cyclooxygenase 2 (COX‐2) product, prostaglandin E₂ (PGE₂), acts through a family of G protein‐coupled receptors designated E‐prostanoid (EP) receptors that mediate intracellular signaling by multiple pathways. However, it is not known whether crosstalk between tumor necrosis factor‐α(TNF‐α)–PGE₂‐mediated signaling and Wnt signaling plays a role in the regulation of intervertebral disc (IVD) cells. In this study, we investigated the relationship between TNF‐α–PGE₂ signaling and Wnt signaling in IVD cells. TNF‐α increased the expression of COX‐2 in IVD cells. The EP receptors EP1, EP3, and EP4 were expressed in IVD cells, and TNF‐α significantly increased PGE₂ production. Stimulation with TNF‐α also upregulated EP3 and EP4 mRNA and protein expression in IVD cells. The inductive effect of the EP3 and EP4 receptors on Topflash promoter activity was confirmed through gain‐ and loss‐of‐function studies using selective EP agonists and antagonists. PGE₂ treatment activated Wnt–β‐catenin signaling through activation of EP3. We conclude that TNF‐α‐induced COX‐2 and PGE₂ stimulate Wnt signaling and activate Wnt target genes. Suppression of the EP3 receptor via TNF‐α–PGE₂ signaling seems to suppress IVD degeneration by controlling the activation of Wnt signaling. These findings may help identify the underlying mechanism and role of Wnt signaling in IVD degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1756–1768, 2015.     

Development of a tissue-engineered human oral mucosa equivalent based on an acellular allogeneic dermal matrix: A preliminary report of clinical application to burn wounds

Tissue-engineered skin equivalents composed of epidermal and dermal components have been widely investigated for coverage of full-thickness skin defects. We developed a tissue-engineered oral mucosa equivalent based on an acellular allogeneic dermal matrix and investigated its characteristics. We also tried and assessed its preliminary clinical application. Human oral mucosal keratinocytes were separated from a piece of oral mucosa and cultured in a chemically-defined medium. The keratinocytes were seeded on to the acellular allogeneic dermal matrix and cultured. Histologically, the mucosa equivalent had a well-stratified epithelial layer. Immunohistochemical study showed that it was similar to normal oral mucosa. We applied this equivalent in one case with an extensive burn wound. The equivalent was transplanted three weeks after the harvest of the patient's oral mucosa and about 30% of the graft finally survived. We conclude that this new oral mucosa equivalent could become a therapeutic option for the treatment of extensive burns.     

Clinical,Biochemical, and Genetic Features of 41 Han Chinese Families With Primary Hypertrophic Osteoarthropathy,and Their Therapeutic Response to Etoricoxib: Results From a Six‐Month Prospective Clinical Intervention

Primary hypertrophic osteoarthropathy (PHO) is a rare inherited disease caused by genetic defects in the prostaglandin metabolism pathway; disturbed prostaglandin E₂ (PGE₂) catabolism resulting in increased PGE₂ level is suggested in the pathogenesis. Forty‐three Han Chinese patients with PHO were studied and 41 of them were treated. Mutations in the HPGD gene, causing hypertrophic osteoarthropathy, primary, autosomal recessive 1 (PHOAR1; OMIM 259100), were identified in seven patients, and mutations in the SLCO2A1 gene, causing hypertrophic osteoarthropathy, primary, autosomal recessive 2 (PHOAR2; OMIM 614441), were identified in 36 patients. Clinical phenotypes of PHO varied, ranging from mild isolated finger clubbing to severe pachydermia and disabling joint swelling, even within families. Circulating PGE₂ metabolism features of PHOAR2 were different from those of PHOAR1. Different frequency and severity of pachydermia between the subgroups were also indicated. A percentage of PHOAR2 patients suffered from gastrointestinal hemorrhage, but this symptom was not observed in the PHOAR1 subgroup. Clinical evidence highlighted the essential role of sex hormones in prostaglandin transporter regulation with respect to PHOAR2 onset, although no significant associations of urinary PGE₂ or PGE‐M with sex hormones were identified. Treatment with etoricoxib, a selective cyclooxygenase‐2 inhibitor, was proved to be beneficial and safe. We detected its notable efficacy in decreasing urinary PGE₂ levels in the majority of the enrolled patients during 6 months of intervention; clinical phenotypes assessed, including pachydermia, finger clubbing, and joint swelling, were improved. We found no visible evidence of a positive effect of etoricoxib on periostosis; however, significant links between urinary PGE₂ and serum bone turnover markers indicated a potential role of decreased PGE₂ in periostosis management. This is the largest reported cohort of subjects genetically diagnosed with PHO. For the first time, we systematically investigated the biochemical and clinical differences between PHOAR1 and PHOAR2, and prospectively showed the positive efficacy and safety of etoricoxib for PHO patients. © 2017 American Society for Bone and Mineral Research.     

Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold

Our aim of this study was to develop a new methodology for constructing a bilayer human skin equivalent to create a more clinical compliance skin graft composite for the treatment of various skin defects. We utilized human plasma derived fibrin as the scaffold for the development of a living bilayer human skin equivalent: fibrin-fibroblast and fibrin-keratinocyte (B-FF/FK SE). Skin cells from six consented patients were culture-expanded to passage 1. For B-FF/FK SE formation, human fibroblasts were embedded in human fibrin matrix and subsequently another layer of human keratinocytes in human fibrin matrix was stacked on top. The B-FF/FK SE was then transplanted to athymic mice model for 4 weeks to evaluate its regeneration and clinical performance. The in vivo B-FF/FK SE has similar properties as native human skin by histological analysis and expression of basal Keratin 14 gene in the epidermal layer and Collagen type I gene in the dermal layer. Electron microscopy analysis of in vivo B-FF/FK SE showed well-formed and continuous epidermal-dermal junction. We have successfully developed a technique to engineer living bilayer human skin equivalent using human fibrin matrix. The utilization of culture-expanded human skin cells and fibrin matrix from human blood will allow a fully autologous human skin equivalent construction.     

Human De-epidermized Dermis as a Stem Cell Carrier

Recently several types of skin equivalents have been developed, consisting of differentiated keratinocytes cultured on various dermal substitutes. Different models of reconstructed human skin have been proposed, such as human and animal de-epidermized dermis, inert filters, collagen matrices, lyophilized collagen membranes populated with fibroblasts, and other models populated with melanocytes and/or Langerhans cells. These skin equivalents mimic native skin in vivo. They have provided information about dermal-epidermal interactions, cell-cell, and cell-matrix interactions; responses of dermal and epithelial cells to biological signals and pharmacological agents; as well as effects of drugs and growth factors on wound healing. Human allodermis from tissue banks has been used for clinical purposes, namely, as support for autologous keratinocyte cultures and as a potentially ideal scaffold for dermal replacement. This bioproduct is considered to be the most suitable clinical carrier for autologous fibroblasts and keratinocytes, as well as an useful experimental model to study angiogenesis and to stimulate vascularization in reconstructed human skin. Because it is human-derived, it is in our opinion the safest of all available types of skin equivalent. Having epidermal and dermal structures, it can be used in one-stage grafting procedures for wound closure.