Exogenous metallothionein‐IIA promotes accelerated healing after a burn wound

Severe injury to the epidermal barrier often results in scarring and life‐long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn₇‐MT‐IIA), a naturally occurring small cysteine‐rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT‐I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT‐I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn₇‐MT‐IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7‐MT‐IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn₇‐MT‐IIA (2 μg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate‐buffered saline (PBS), 8.9±0.3 mm diameter vs. MT‐I/II, 7.1±0.7 mm; 7 days: PBS 5.8±0.98 mm vs. MT‐I/II, 3.6±1.0 mm, p<0.05) and reduced epidermal thickness (MT‐I/II: 45±4 μm vs. PBS: 101±19 μm, p<0.05) compared with controls. Our data suggest that exogenous Zn₇‐MT‐IIA may prove a valuable therapeutic for patients with burns and other skin injuries.     

Skin cells, but not T cells, are resistant to indoleamine 2, 3-dioxygenase (IDO) expressed by allogeneic fibroblasts

We have previously demonstrated that indoleamine 2, 3‐dioxygenase (IDO) expressed by dermal fibroblasts generated a tryptophan deficient environment in which immune cells, but not skin cells, undergo apoptosis. However, the mechanism by which primary skin cells such as fibroblasts and keratinocytes are resistant to this culture environment is not elucidated. Here, we asked the question of whether the activity of the general control nonderepressing‐2 (GCN2) kinase pathway in primary immune and skin cells is differently regulated in response to IDO‐induced tryptophan deficient environment. Before addressing this question, the expression of IDO in IDO‐adenoviral infected fibroblasts, as a source of IDO expression, was validated. We then demonstrated a significant immunosuppressive effect of IDO expression in primary human T cells co‐cultured with IDO expressing fibroblasts in the presence of allogeneic pieces of either epidermis or full thickness skin. Evaluating the mechanism by which skin cells, but not T cells, are resistant to IDO induced low tryptophan environment, we then co‐cultured IDO‐expressing fibroblasts with bystander human T cells, the fibroblasts, or keratinocytes for 3 days. The results showed a significant activation of apoptotic pathway as analyzed by caspase‐3 induction as well as the expression of CHOP, a downstream effector of GCN2 kinase pathway in T cells, but not in skin cells.     

Therapeutic efficacy of autologous platelet‐rich plasma and polydeoxyribonucleotide on female pattern hair loss

Autologous platelet‐rich plasma (PRP) exerts positive therapeutic effects on hair thickness and density in patients with pattern hair loss. The aim of our study was to evaluate the efficacy of intra‐perifollicular autologous PRP and polydeoxyribonucleotide (PDRN) injections in treating female pattern hair loss (FPHL). Twenty FPHL patients were treated with a single session of PRP injection, followed by 12 sessions of PDRN intra‐perifollicular injection, along the scalp at weekly intervals. Additionally, another 20 FPHL patients were treated with 12 sessions of PDRN injection only. Meanwhile, one half of the backs of two rabbits was injected with the PRP preparation, while the other half was injected with phosphate buffered saline as a control. Tissue samples from the rabbits were analyzed by real‐time polymerase chain reaction and Western blotting. Compared with baseline values, patients treated with PRP and PDRN injections exhibited clinical improvement in mean hair counts (23.2 ± 15.5%; p < 0.001) and mean hair thickness (16.8 ± 10.8%; p < 0.001). In addition, patients treated with the 12 sessions of intra‐perifollicular PDRN injection alone also showed clinical improvement in mean hair counts (17.9 ± 13.2%; p < 0.001) and mean hair thickness (13.5 ± 10.7%; p < 0.001). Comparison analyses between the two groups revealed that combined therapy with PRP and PDRN induces greater improvement in hair thickness than treatment with PDRN therapy alone (p = 0.031), but not in hair counts (p > 0.05). The pilot animal study revealed significant up‐regulation of WNT, platelet‐derived growth factor, and fibroblast growth factor expression in rabbit skin treated with the PRP preparation, compared with control skin. In conclusion, intra‐perifollicular injections of autologous PRP and/or PDRN generate improvements in hair thickness and density in FPHL patients.     

Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6–9/group) were pulsed with 5‐bromo‐2'‐deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3–6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU‐positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long‐term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue‐engineered skin to natural skin, and contribute to more rapid and stable wound healing.     

Topical application of a film‐forming emulgel dressing that controls the release of stratifin and acetylsalicylic acid and improves/prevents hypertrophic scarring

Here, we evaluate the efficacy of an emulgel dressing to control the release of an antifibrogenic factor, stratifin (SFN), along with an anti‐inflammatory drug, acetylsalicylic acid (ASA), to be used as a wound dressing with hypertrophic scar reducing features. Emulgel dressings were prepared by dispersing positively charged submicron vesicles in carboxymethyl cellulose gel. Release kinetics of SFN/ASA and toxicity for primary skin cells were assessed in vitro. Antifibrogenic efficacy of medicated emulgel dressings was tested on a rabbit ear fibrotic model. Following topical application on the wounds, emulgels formed an occlusive film and controlled the release of SFN and ASA for 7 and 24 hours, respectively. Wounds treated with SFN/ASA‐containing emulgel dressings showed an 80% reduction in scar elevation compared with untreated controls. Topical formulations were nontoxic for cultured human keratinocytes and fibroblasts. Inflammation was significantly controlled in treated wounds, as shown by a reduced number of infiltrated CD3⁺ T cells (p < 0.001) and macrophages. SFN/ASA‐treated wounds showed a significantly higher (p < 0.001) expression of matrix metalloproteinase‐1, resulting in reduced collagen deposition and less scarring. Film‐forming emulgel dressings that control the release of antifibrogenic and anti‐inflammatory factors provide an excellent treatment option for postburn hypertrophic scar management.     

Comparison of human umbilical cord blood‐derived mesenchymal stem cells with healthy fibroblasts on wound‐healing activity of diabetic fibroblasts

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood‐derived mesenchymal stromal cells (hUCB‐MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB‐MSCs have a superior wound‐healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB‐MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB‐MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB‐MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB‐MSCs. After a 3‐day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB‐MSC‐treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast‐treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB‐MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing.     

Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A

Gene therapy promises the potential for improved treatment of cutaneous wounds. This study evaluated whether genetically modified cultured skin substitutes can act as vehicles for gene therapy in an athymic mouse model of wound healing. Human keratinocytes and fibroblasts were genetically engineered by retroviral transduction to overexpress human platelet-derived growth factor-A chain. Three types of skin substitutes were prepared from collagen-glycosaminoglycan substrates populated with fibroblasts and keratinocytes: HF-/HK-, containing both unmodified fibroblasts and keratinocytes; HF-/HK+, containing unmodified fibroblasts and modified keratinocytes; and HF+/HK-, containing modified fibroblasts and unmodified keratinocytes. Skin substitutes were cultured for two weeks before grafting to full-thickness wounds on athymic mice. The modified skin substitutes secreted significantly elevated levels of platelet-derived growth factor throughout the culture period. Expression of retroviral platelet-derived growth factor-A mRNA was maintained after grafting to mice, and was detected in all HF-/HK+ grafts and one HF+/HK- graft at two weeks after surgery. Although no differences were seen between control and modified grafts, the results suggest that genetically modified cultured skin substitutes can be a feasible mechanism for cutaneous gene therapy. The cultured skin model used for these studies has advantages over other skin analogs containing only epidermal cells; because it contains both fibroblasts and keratinocytes, it therefore offers greater opportunities for genetic modification and potential modulation of wound healing.     

Proliferation of peripheral blood mononuclear cells is suppressed by the indoleamine 2,3-dioxygenase expression of interferon-γ–treated skin cells in a co-culture system

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme possessing various immunosuppressive characteristics. In this study, we report the possible use of this enzyme in an allogenic skin substitute to suppress the proliferation of immune cells. Human fetal skin fibroblasts and keratinocytes were treated with the cytokine interferon-gamma to induce expression of IDO mRNA and protein. IDO enzyme activity was evaluated by measurement of kynurenine levels in the interferon-gamma-treated and -untreated cells. Results of Northern analysis showed a dose-dependent response in expression of IDO mRNA to the various concentrations of interferon-gamma used. Northern blot analysis also showed a time-dependent expression of IDO in response to different durations of interferon-gamma treatment. The level of kynurenine measured, as the bioactivity of IDO enzyme, was significantly higher in the interferon-gamma-treated fibroblasts and keratinocytes compared to those of controls (p < 0.001). To illustrate the immunosuppressive effects of IDO on immune cell proliferation, IDO-expressing fibroblasts were cocultured with human peripheral blood mononuclear cells for a period of 5 days. Results of 3H-thymidine incorporation assays showed a significant reduction in proliferation of the mononuclear cells cocultured with IDO-expressing skin cells compared to monocytes cocultured with control (non-IDO-expressing) skin cells (p < 0.001). Furthermore, addition of the IDO-inhibitor (1-methyl-D-tryptophan) significantly reversed the immunosuppressive effects of IDO on monocyte proliferation (p < 0.001). In conclusion, suppression of peripheral blood mononuclear cell proliferation due to interferon-gamma-induced IDO-expression in allogenic human skin cells might shed new light on developing a nonrejectable allogenic skin substitute.     

Differing effects of PTH 1–34, PTH 1–84, and zoledronic acid on bone microarchitecture and estimated strength in postmenopausal women with osteoporosis: An 18‐month open‐labeled observational study using HR‐pQCT

Whereas the beneficial effects of intermittent treatment with parathyroid hormone (PTH) (intact PTH 1–84 or fragment PTH 1–34, teriparatide) on vertebral strength is well documented, treatment may not be equally effective in the peripheral skeleton. We used high‐resolution peripheral quantitative computed tomography (HR‐pQCT) to detail effects on compartmental geometry, density, and microarchitecture as well as finite element (FE) estimated integral strength at the distal radius and tibia in postmenopausal osteoporotic women treated with PTH 1–34 (20 µg sc daily, n = 18) or PTH 1–84 (100 µg sc daily, n = 20) for 18 months in an open‐label, nonrandomized study. A group of postmenopausal osteoporotic women receiving zoledronic acid (5 mg infusion once yearly, n = 33) was also included. Anabolic therapy increased cortical porosity in radius (PTH 1–34 32 ± 37%, PTH 1–84 39 ± 32%, both p < 0.001) and tibia (PTH 1–34 13 ± 27%, PTH 1–84 15 ± 22%, both p < 0.001) with corresponding declines in cortical density. With PTH 1–34, increases in cortical thickness in radius (2.0 ± 3.8%, p < 0.05) and tibia (3.8 ± 10.4%, p < 0.01) were found. Trabecular number increased in tibia with both PTH 1–34 (4.2 ± 7.1%, p < 0.05) and PTH 1–84 (5.3 ± 8.3%, p < 0.01). Zoledronic acid did not impact cortical porosity at either site but increased cortical thickness (3.0 ± 3.5%, p < 0.01), total (2.7 ± 2.5%, p < 0.001) and cortical density (1.5 ± 2.0%, p < 0.01) in tibia as well as trabecular volume fraction in radius (2.5 ± 5.1%, p < 0.05) and tibia (2.2 ± 2.2%, p < 0.01). FE estimated bone strength was preserved, but not increased, with PTH 1–34 and zoledronic acid at both sites, whereas it decreased with PTH 1–84 in radius (?2.8 ± 5.8%, p < 0.05) and tibia (–3.9 ± 4.8%, p < 0.001). Conclusively, divergent treatment‐specific effects in cortical and trabecular bone were observed with anabolic and zoledronic acid therapy. The finding of decreased estimated strength with PTH 1–84 treatment was surprising and warrants confirmation. © 2013 American Society for Bone and Mineral Research.     

Stimulatory effect of fibroblast‐derived prostaglandin E2 on keratinocyte stratification in the skin equivalent

Epidermal–dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin‐1 (IL‐1) and an IL‐1‐induced inflammatory mediator prostaglandin E₂ (PGE₂) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL‐1α mRNA (IL1A mRNA) and relatively low levels of IL‐1β mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin‐endoperoxide synthase 2 mRNA (PTGS2 mRNA) and production of PGE₂ in three‐dimensionally cultured fibroblasts were noticeably stimulated by co‐culture with keratinocytes, whereas PTGS2 mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15‐(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL‐1β stimulated PTGS2 mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67‐positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE₂ was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL‐1 and PGE₂.     

Endotoxin‐induced inflammation in a rodent model up‐regulates IL‐1a expression and CD45+ leukocyte recruitment and increases the rate of reepithelialization and wound closure

Wound healing is traditionally divided into inflammation, proliferation, and remodeling phases. Several inflammatory mediators and cells regulate the inflammation phase. The specific roles for different mediators have not been clearly defined. The effects of inflammation phase modulation on wound healing were evaluated in this study. Rat full‐thickness wounds were divided into different experimental groups: (1) sterile hyper‐inflammatory wounds/endotoxin (topical endotoxin), (2) sterile hypo‐inflammatory/inhibitor group (cocktail of topical COX‐1 plus COX‐2 plus lipoxygenase inhibitors), and (3) control groups: topical saline or DMSO. After full‐thickness wound creation, custom‐made titanium chambers enclosed the wound, creating an isolated well‐controlled environment. Wound healing was followed over time; tissue biopsies and wound fluid samples were collected on days 1, 4, and 8 postoperatively. The validity of the inflammation model was confirmed by increased IL‐1a expression, increased CD45+ leukocytes recruitment in the hyper‐inflamed group as compared to the inhibitor and control groups. The reepithelialization percentage was significantly increased in the endotoxin group as compared to the inhibitor group on day 4 (60.75 vs. 22.05, p‐value <0.05) and both the inhibitor and the control group on day 8 (control group: 63.2%, inhibitor group: 28.9%, endotoxin group: 84.2%, p‐value <0.05). Also, the macroscopic wound closure was increased in the endotoxin group as compared to the inhibitor group and control group both on day 4 (control group: 69.9%, inhibitor group: 62.9%, endotoxin group: 81.9%, p‐value <0.05) and on day 8 (control group: 68.5%, inhibitor group: 69.1%, endotoxin group: 83.7%, p‐value <0.05). Endotoxin‐induced sterile inflammation up‐regulates IL‐1a expression and CD45+ leukocyte recruitment and results in faster rate of wound reepithelialization and wound closure in full‐thickness rodent wounds. Conversely, the wound reepithelialization and wound closure can be significantly delayed on treatment with a combination of cyclooxygenase and lipoxygenase inhibitors.     

Constitutive expression of hypoxia‐inducible factor‐1 α in keratinocytes during the repair of skin wounds in horses

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia‐inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin‐dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia‐independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.     

Basic fibroblast growth factor accelerates and improves second‐degree burn wound healing

Second‐degree burns are sometimes a concern for shortening patient suffering time as well as the therapeutic choice. Thus, adult second‐degree burn patients (average 57.8 ± 13.9 years old), mainly with deep dermal burns, were included. Patients receiving topical basic fibroblast growth factor (bFGF) or no bFGF were compared for clinical scar extent, passive scar hardness and elasticity using a Cutometer, direct scar hardness using a durometer, and moisture analysis of the stratum corneum at 1 year after complete wound healing. There was significantly faster wound healing with bFGF, as early as 2.2 ± 0.9 days from the burn injury, compared with non‐bFGF use (12.0 ± 2.2 vs. 15.0 ± 2.7 days, p<0.01). Clinical evaluation of Vancouver scale scores showed significant differences between bFGF‐treated and non‐bFGF–treated scars (p<0.01). Both maximal scar extension and the ratio of scar retraction to maximal scar extension, elasticity, by Cutometer were significantly greater in bFGF‐treated scars than non‐bFGF–treated scars (0.23 ± 0.10 vs. 0.14 ± 0.06 mm, 0.59 ± 0.20 vs. 0.49 ± 0.15 mm: scar extension, scar elasticity, bFGF vs. non‐bFGF, p<0.01). The durometer reading was significantly lower in bFGF‐treated scars than in non‐bFGF–treated scars (16.2 ± 3.8 vs. 29.3 ± 5.1, p<0.01). Transepidermal water loss, water content, and corneal thickness were significantly less in bFGF‐treated than in non‐bFGF–treated scars (p<0.01).     

Treatment of chronic diabetic lower leg ulcers with activated protein C: a randomised placebo‐controlled,double‐blind pilot clinical trial

Lower leg ulcers are a serious and long‐term complication in patients with diabetes and pose a major health concern because of the increasing number of patients diagnosed with diabetes each year. This study sought to evaluate the clinical benefit of topical activated protein C (APC) on chronic lower leg ulcers in patients with diabetes. Twelve patients were randomly assigned to receive either APC (N = 6) or physiological saline (placebo; N = 6) in a randomised, placebo‐controlled, double‐blind pilot clinical trial. Treatment was administered topically, twice weekly for 6 weeks with final follow‐up at 20 weeks. Wound area was significantly reduced to 34·8 ± 16·4% of week 0 levels at 20 weeks in APC‐treated wounds (p = 0·01). At 20 weeks, three APC‐treated wounds had completely healed, compared to one saline‐treated wound. Full‐thickness wound edge skin biopsies showed reduced inflammatory cell infiltration and increased vascular proliferation following APC treatment. Patient stress scores were also significantly reduced following APC treatment (p < 0·05), demonstrating improved patient quality of life as assessed by the Cardiff Wound Impact Questionnaire. This pilot trial suggests that APC is a safe topical agent for healing chronic lower leg ulcers in patients with diabetes and provides supporting evidence for a larger clinical trial.     

Adipose‐derived aldehyde dehydrogenase–expressing cells promote dermal regenerative potential with collagen‐glycosaminoglycan scaffold

Aldehyde dehydrogenase (ALDH) is an enzyme that plays an important role in retinoid metabolism and highly expressed in stem cells. This study isolated ALDH‐expressing cells from subcutaneous adipose tissue and investigated their potential to enhance healing in a full‐thickness skin wound in rats by co‐implanting them with collagen‐glycosaminoglycan (c‐GAG) scaffolds. ALDH‐positive cells were isolated by a fluorescence‐activated cell sorting technique from Lewis rat's stromal‐vascular‐fraction (SVF) and transplanted with c‐GAG scaffolds in a rat full‐thickness skin wound model. At 7 days after surgery, the microscopic appearance of c‐GAG scaffolds seeded with ALDH‐positive was compared with those of uncultured‐SVF, and cultured‐SVF adipose‐derived stromal cells (ASCs). The thickness of cellular ingrowth in the ASC group (630 ± 180 μm) was significantly thicker than that in the control (390 ± 120 μm) or SVF (380 ± 140 μm) groups, but non‐significantly thicker than that in the ALDH‐positive group (570 ± 220 μm). The thickness of regenerated collagen layer was significantly thicker in the ALDH‐positive group (160 ± 110 μm) than in the ASCs (81 ± 41 μm), the control (65 ± 24 μm), or SVF (64 ± 34 μm) groups. Immunofluorescent staining with CD31 proved that transplanted ALDH‐positive cells differentiated into vascular endothelial cells in c‐GAG scaffolds. Combined transplantation with c‐GAG scaffolds and adipose‐derived ALDH‐positive cells promoted dermal regeneration, giving a possibility that ALDH‐positive cells would greatly shorten the waiting period before secondary autologous skin grafting was possible.     

Low‐magnitude high‐frequency vibration enhances gene expression related to callus formation,mineralization and remodeling during osteoporotic fracture healing in rats

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col‐2), osteogenesis (Col‐1) and remodeling (receptor activator for nuclear factor‐ κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post‐treatment. LMHFV enhanced osteoporotic fracture healing by up‐regulating the expression of chondrogenesis‐, osteogenesis‐ and remodeling‐related genes (Col‐2 at week 4 (p = 0.008), Col‐1 at week 2 and 8 (p < 0.001and p = 0.008) and RANKL/OPG at week 8 (p = 0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col‐2 and Col‐1 at week 2 (p < 0.001 for all), larger callus width at week 2 (p = 0.001), callus area at week 1 and 5(p < 0.05 for all) and greater relative area of osseous tissue (p = 0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1572–1579, 2014.     

Skin substitute‐assisted repair shows reduced dermal fibrosis in acute human wounds validated simultaneously by histology and optical coherence tomography

Skin substitutes are heterogeneous biomaterials designed to accelerate wound healing through provision of replacement extracellular matrix. Despite growing evidence for their use in chronic wounds, the role of skin substitutes in acute wound management and their influence on fibrogenesis remains unclear. Skin substitute characteristics including biocompatibility, porosity, and elasticity strongly influence cellular behavior during wound healing. Thus, we hypothesize that structural and biomechanical variation between biomaterials may induce differential scar formation after cutaneous injury. The following human prospective cohort study was designed to investigate this premise. Four 5‐mm full thickness punch biopsies were harvested from 50 volunteers. In all cases, site 1 healed by secondary intention, site 2 was treated with collagen‐GAG scaffold (CG), and decellularised dermis (DCD) was applied to site 3 while tissue extracted from site 4 was replaced (autograft). Healing tissue was assessed weekly with optical coherence tomography (OCT), before being excised on days 7, 14, 21, or 28 depending on study group allocation for later histological and immunohistochemical evaluation. Extracted RNA was used in microarray analysis and polymerase chain reaction of highlighted genes. Autograft treatment resulted in minimal fibrosis confirmed immunohistochemically and with OCT through significantly lower collagen I levels (p = 0.047 and 0.03) and reduced mean grayscale values (p = 0.038 and 0.015), respectively. DCD developed intermediate scar formation with partial rete ridge reformation and reduced fasiculonodular fibrosis. It was uniquely associated with late up‐regulation of matrix metalloproteinases 1 and 3, oncostatin M, and interleukin‐10 (p = 0.007, 0.04, 0.019, 0.019). Regenerated dermis was significantly thicker in DCD and autografts 28 days post‐injury compared with control and CG samples (p = 0.003 and <0.0001). In conclusion, variable fibrotic outcomes were observed in skin substitute‐treated wounds with reduced scarring in autograft and DCD samples compared with controls. OCT enabled concurrent assessment of wound morphology and quantification of dermal fibrosis.     

In vivo methods to evaluate a new skin protectant for loss of skin integrity

A new skin protectant was developed for use on conditions involving partial‐thickness skin loss such as severe incontinence‐associated dermatitis. This new formulation is based on a cyanoacrylate chemistry designed to polymerize in situ and create a breathable film able to protect the skin surface from external irritants. This film provides an environment favorable for healing to occur beneath the film. To evaluate the characteristics of the novel chemistry, we devised a preclinical testing strategy comprising three different animal models. The data from all three models was considered collectively to create an overall assessment of effectiveness. A guinea pig model was used to evaluate the barrier efficacy of the new product in protecting intact skin from irritation. A porcine partial‐thickness wound model was used to evaluate the efficacy of the product in helping control minor bleeding and exudate. A similar model was also used to assess the process of reepithelialization in the continued presence of an irritant. In the first model, untreated sites had 8.5 times more irritation than sites covered with the new product (p < 0.001). In the second model, a single application of the new product successfully attached to intact peri‐wound skin and to denuded, weepy skin. It significantly reduced the amount of fluid weeping from the wounds (p ≤ 0.001) and continued to perform throughout a 96 hours experiment. In the third model, the percent of reepithelialization was significantly greater for the wounds covered with the new product than for the control wounds (p = 0.003; on average, 18.3% greater, with a 95% confidence interval of 9.2% to 27.5%). These results suggest that the new skin protectant protects intact and denuded skin from irritants and provides an environment favorable to healing, offering promise for the management of various conditions involving loss of epidermis.     

Deleterious Effect of Late Menarche on Distal Tibia Microstructure in Healthy 20‐Year‐Old and Premenopausal Middle‐Aged Women

Late menarche is a risk factor for fragility fractures. We hypothesized that pubertal timing–dependent alterations in bone structural components would persist from peak bone mass to menopause, independent of premenopausal bone loss. We studied the influence of menarcheal age (MENA) on femoral neck BMD (FN aBMD) by DXA and microstructure of distal tibia by HR‐pQCT in healthy young adult (YAD; 20.4 ± 0.6 [SD] yr, n = 124) and premenopausal middle‐aged (PREMENO; 45.8 ± 3.4 yr, n = 120) women. Median of MENA was 13.0 ± 1.2 and 13.1 ± 1.7 yr in YAD and PREMENO, respectively. In YAD and PREMENO (n = 244), FN aBMD (R = ?0.29, p = 0.013), as well as total volumetric BMD (Dtot; R = ?0.23, p = 0.006) and cortical thickness (Ct.Th; R = ?0.18, p = 0.011) of distal tibia were inversely correlated to MENA. After segregation by the median of MENA in EARLY and LATE subgroups, the significant influences of both MENA (p = 0.004) and chronological age (p < 0.0001) were observed for FN aBMD and trabecular bone volume fraction of the distal tibia with similar differences in T‐scores between LATE and EARLY subgroups in YAD (?0.36 and ?0.31 T‐scores) and PREMENO (?0.35 and ?0.42 T‐scores) women. Ct.Th was negatively influenced by MENA, whereas trabecular thickness (Tb.Th) was negatively influenced by chronological age. There was a striking inverse relationship between cross‐sectional area and Ct.Th (R = ?0.57, p < 0.001). In conclusion, the negative influence of late menarcheal age at weight‐bearing sites as observed by the end of skeletal growth remains unattenuated a few years before menopause and is independent of premenopausal bone loss. Alterations in both bone mineral mass and microstructural components may explain the increased risk of fragility fractures associated with later menarcheal age.