Growth hormone‐releasing peptide 6 prevents cutaneous hypertrophic scarring: early mechanistic data from a proteome study

Hypertrophic scars (HTS) and keloids are forms of aberrant cutaneous healing with excessive extracellular matrix (ECM) deposition. Current therapies still fall short and cause undesired effects. We aimed to thoroughly evaluate the ability of growth hormone releasing peptide 6 (GHRP6) to both prevent and reverse cutaneous fibrosis and to acquire the earliest proteome data supporting GHRP6's acute impact on aesthetic wound healing. Two independent sets of experiments addressing prevention and reversion effects were conducted on the classic HTS model in rabbits. In the prevention approach, the wounds were assigned to topically receive GHRP6, triamcinolone acetonide (TA), or vehicle (1% sodium carboxy methylcellulose [CMC]) from day 1 to day 30 post‐wounding. The reversion scheme was based on the infiltration of either GHRP6 or sterile saline in mature HTS for 4 consecutive weeks. The incidence and appearance of HTS were systematically monitored. The sub‐epidermal fibrotic core area of HTS was ultrasonographically determined, and the scar elevation index was calculated on haematoxylin/eosin‐stained, microscopic digitised images. Tissue samples were collected for proteomics after 1 hour of HTS induction and treatment with either GHRP6 or vehicle. GHRP6 prevented the onset of HTS without the untoward reactions induced by the first‐line treatment triamcinolone acetonide (TA); however, it failed to significantly reverse mature HTS. The preliminary proteomic study suggests that the anti‐fibrotic preventing effect exerted by GHRP6 depends on different pathways involved in lipid metabolism, cytoskeleton arrangements, epidermal cells’ differentiation, and ECM dynamics. These results enlighten the potential success of GHRP6 as one of the incoming alternatives for HTS prevention.     

Improvement of hypertrophic scarring by using topical anti‐fibrogenic/anti‐inflammatory factors in a rabbit ear model

This study investigates the scar‐reducing efficacy of topical application of stratifin and acetylsalicylic acid (ASA) in a rabbit ear model. A total of five New Zealand white rabbits with four wounds per ear were examined. Either recombinant stratifin (0.002%) or ASA (0.5%) incorporated in carboxymethyl cellulose gel was topically applied on each wound at postwounding Day 5. Scars were harvested at postwounding Day 28 for histological analysis. The wounds treated with stratifin and ASA showed 82 and 73% reduction in scar volume, respectively, compared with that of untreated controls. A reduction of 57 and 41% in total tissue cellularity along with 79 and 91% reduction in infiltrated CD3⁺ T cells were observed in response to treatment with stratifin and ASA, respectively, compared with those of untreated controls. Wounds treated with stratifin showed a 2.8‐fold increase in matrix metalloproteinase‐1 expression, which resulted in a 48% decrease in collagen density compared with those of untreated controls. Qualitative wound assessment showed a reduced hypertrophic scarring in stratifin and ASA‐treated wounds when compared with the controls. This study showed that topical application of either stratifin or ASA‐impregnated carboxymethyl cellulose gel reduced hypertrophic scar formation following dermal injuries in a rabbit ear fibrotic model.     

The therapeutic potential of a C‐X‐C chemokine receptor type 4 (CXCR‐4) antagonist on hypertrophic scarring in vivo

Effective prevention and treatment of hypertrophic scars (HTSs), a dermal form of fibrosis that frequently occurs following thermal injury to deep dermis, are unsolved significant clinical problems. Previously, we have found that stromal cell‐derived factor 1/CXCR4 signaling is up‐regulated during wound healing in burn patients and HTS tissue after thermal injury. We hypothesize that blood‐borne mononuclear cells are recruited into wound sites after burn injury through the chemokine pathway of stromal cell‐derived factor 1 and its receptor CXCR4. Deep dermal injuries to the skin are often accompanied by prolonged inflammation, which leads to chemotaxis of mononuclear cells into the wounds by chemokine signaling where fibroblast activation occurs and ultimately HTS are formed. Blocking mononuclear cell recruitment and fibroblast activation, CXCR4 antagonism is expected to reduce or minimize scar formation. In this study, the inhibitory effect of CXCR4 antagonist CTCE‐9908 on dermal fibrosis was determined in vivo using a human HTS‐like nude mouse model, in which split‐thickness human skin is transplanted into full‐thickness dorsal excisional wounds in athymic mice, where these wounds subsequently develop fibrotic scars that resemble human HTS as previously described. CTCE‐9908 significantly attenuated scar formation and contraction, reduced the accumulation of macrophages and myofibroblasts, enhanced the remodeling of collagen fibers, and down‐regulated the gene and protein expression of fibrotic growth factors in the human skin tissues. These findings support the potential therapeutic value of CXCR4 antagonist in dermal fibrosis and possibly other fibroproliferative disorders.     

Topical application of recombinant platelet‐derived growth factor increases the rate of healing and the level of proteins that regulate this response

A bipedicle ischaemic rat skin flap model was used to study the effects of daily topical applications of platelet‐derived growth factor (PDGF) on the healing of ischaemic wounds. Levels of tumour necrosis factor‐alpha (TNFA), interleukin 1‐beta (IL1B) and both the latent and active forms of matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were measured. Full‐thickness wounds were made on a total of 72 adult male Sprague–Dawley rats. Each group of 18 rats with normal and ischaemic wounds received either vehicle or 0·01% recombinant PDGF‐BB. Additional applications were made on the wounds on a daily basis. Wound areas were measured at 0, 1, 3, 5, 7 9 and 13 days after wounding. Ischaemia caused a delay in wound healing as well as an increase in TNFA, IL1B and both the pro and active forms of MMP2 and MMP9. PDGF accelerated the rate of wound healing in both normal and ischaemic wounds and negated the effect of ischaemia. PDGF reduced the TNFA concentration in both normal and ischaemic wounds, and the rate of wound healing closely resembled the pattern of TNFA protein expression. PDGF also reduced both the magnitude and duration of the increases in IL1B and both the pro and active forms of MMP2 and MMP9 induced by ischaemia.     

Treatment with acetylsalicylic acid prevents short to mid-term radiographic progression of nontraumatic osteonecrosis of the femoral head: a pilot study

Background

Nontraumatic osteonecrosis of the femoral head (ONFH) is a progressive disease in young adults producing substantial morbidity and frequently resulting in total hip arthroplasty. Although hip-preserving surgical procedures represent the current mainstay of treatment for early disease, medical therapies targeting specific pathways in the ONFH pathogenesis could help prevent disease progression while producing less morbidity. Acetylsalicylic acid (ASA) is a promising alternative to other therapies for ONFH owing to its anti-inflammatory and antithrombotic mechanisms of action and its relatively benign side effect profile.

Methods

We followed a prospective cohort of 10 patients (12 hips) with precollapse ONFH who were given ASA to prevent disease progression. Their outcomes were compared with those of a historic control group taken from the literature.

Results

Progression occurred in 1 of 12 (8%) patients taking ASA compared with 30 of 45 (66.6%) controls (p = 0.002) at a mean follow-up of 3.7 years. Patients taking ASA also tended to exhibit decreased femoral head involvement at the end of therapy.

Conclusion

This hypothesis-generating study leads us to believe that ASA may be a simple and effective treatment option for delaying disease progression in patients with early-stage ONFH.     

Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury

In postoperative patients with head and neck cancer, scar tissue formation may interfere with the healing process, resulting in incomplete functional recovery and a reduced quality of life. Percutaneous application of carbon dioxide (CO₂) has been reported to improve hypoxia, stimulate angiogenesis, and promote fracture repair and muscle damage. However, gaseous CO₂ cannot be applied to the head and neck regions. Previously, we developed a paste that holds non-gaseous CO₂ in a carrier and can be administered transdermally. Here, we investigated whether this paste could prevent excessive scarring and promote muscle regeneration using a bupivacaine-induced rat model of muscle injury. Forty-eight Sprague Dawley rats were randomly assigned to either a control group or a CO₂ group. Both groups underwent surgery to induce muscle injury, but the control group received no treatment, whereas the CO₂ group received the CO₂ paste daily after surgery. Then, samples of the experimental sites were taken on days 3, 7, 14, and 21 post-surgery to examine the following: (1) inflammatory (interleukin [IL]-1β, IL-6), and transforming growth factor (TGF)-β and myogenic (MyoD and myogenin) gene expression by polymerase chain reaction, (2) muscle regeneration with haematoxylin and eosin staining, and (3) MyoD and myogenin protein expression using immunohistochemical staining. Rats in the CO₂ group showed higher MyoD and myogenin expression and lower IL-1β, IL-6, and TGF-β expression than the control rats. In addition, treated rats showed evidence of accelerated muscle regeneration. Our study demonstrated that the CO₂ paste prevents excessive scarring and accelerates muscle regeneration. This action may be exerted through the induction of an artificial Bohr effect, which leads to the upregulation of MyoD and myogenin, and the downregulation of IL-1β, IL-6, and TGF-β. The paste is inexpensive and non-invasive. Thus, it may be the treatment of choice for patients with muscle damage.     

One intra‐articular injection of hyaluronan prevents cell death and improves cell metabolism in a model of injured articular cartilage in the rabbit

The purpose of this study was to determine the effect of one intra‐articular injection of hyaluronan on chondrocyte death and metabolism in injured cartilage. Twenty‐three 6‐month‐old rabbits received partial‐thickness articular cartilage defects created on each medial femoral condyle. In order to examine the effect on articular cartilage surrounding iatrogenic cartilage lesions, which can occur during arthroscopic procedures, Study 1 was performed: in 14 rabbits both knees were immediately rinsed with 0.9% NaCl. Experimental knees were treated with hyaluronan. Six rabbits were sacrificed at 2 days; eight rabbits 3 months postoperatively. Histomorphometric analysis was used for studying cell death in cartilage next to the defect. In order to examine the effect on longer lasting lesions, more reflecting the clinical situation, Study 2 was performed: after 6 months knee joints of nine rabbits were (i) irrigated with 0.9% NaCl, (ii) treated with hyaluronan after irrigation with 0.9% NaCl, or (iii) sham‐treated. After 7 days patellas were used to study the chondrocyte metabolism by measuring the [³⁵S]sulfate incorporation. Study 1: Two days postoperatively, in hyaluronan‐treated cartilage the percentage of dead cells was 6.7%, which was significantly lower compared to 16.2% in saline‐treated cartilage. After 3 months the percentages of dead cells in both groups were statistically similar. Study 2: Hyaluronan treatment resulted in significantly higher [³⁵S]sulfate incorporation compared to knees irrigated with 0.9% NaCl. These results suggest a potential role for hyaluronan in preventing cell death following articular cartilage injury. One injection of hyaluronan improved cartilage metabolism in knees with 6‐month‐old cartilage defects. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:624–630, 2008     

Enhanced wound‐healing performance of a phyto‐polysaccharide‐enriched dressing – a preclinical small and large animal study

Alginate is a natural rich anionic polysaccharide (APS), commonly available as calcium alginate (CAPS). It can maintain a physiologically moist microenvironment, which minimises bacterial infection and facilitates wound healing at a wound site. Patients with burn injuries suffer from pain and an inflammatory response. In this study, we evaluated the CAPS dressing and traditional dressing containing carboxymethyl cellulose (CMC) for wound healing and scar tissue formation in a burn model of rat and swine. In our pilot study of a burn rat model to evaluate inflammatory response and wound healing, we found that the monocyte chemoattractant protein (MCP)‐1 and transforming growth factor (TGF)‐β were up‐regulated in the CAPS treatment group. Next, the burn swine models tested positive for MCP‐1 in a Gram‐positive bacterial infection, and there was overproduction of TGF‐β during the burn wound healing process. Rats were monitored daily for 1 week for cytokine assay and sacrificed on day 28 post‐burn injury. The swine were monitored over 6 weeks. We further examined the pain and related factors and inflammatory cytokine expression in a rodent burns model monitored everyday for 7 days post‐burn. Our results revealed that the efficacy of the dressing containing CAPS for wound repair post‐burn was better than the CMC dressing with respect to natural wound healing and scar formation. The polysaccharide‐enriched dressing exerted an antimicrobial effect on burn wounds, regulated the inflammatory response and stimulated anti‐inflammatory cytokine release. However, one pain assessment method showed no significant difference in the reduction in levels of adenosine triphosphate in serum of rats after wound dressing in either the CAPS or CMC group. In conclusion, a polysaccharide‐enriched dressing outperformed a traditional dressing in reducing wound size, minimising hypertrophic scar formation, regulating cytokines and maximising antimicrobial effects.     

Topical application of 17β‐estradiol (E2) improves skin flap survival through activation of endothelial nitric oxide synthase in rats

This study investigates the influence of 17β‐estradiol (E2) on nitric oxide (NO) production in endothelial cell cultures and the effect of topical E2 on the survival of skin flap transplants in a rat model. Human umbilical vein endothelial cells were treated with three different E2 concentrations and nitrite (NO ₂) concentrations, as well as endothelial nitric oxide synthase (eNOS) protein expressions were analyzed. In vivo, random‐pattern skin flaps were raised in female Wistar rats 14 days following ovariectomy and treated with placebo ointment (group 1), E2 as gel (group 2), and E2 via plaster (group 3). Flap perfusion, survival, and NO ₂ levels were measured on postoperative day 7. In vitro, E2 treatment increased NO ₂ concentration in cell supernatant and eNOS expression in cell lysates (p < 0.05). In vivo, E2 treated (gel and plaster groups) demonstrated significantly increased skin flap survival compared to the placebo group (p < 0.05). E2 plaster‐treated animals exhibited higher NO ₂ blood levels than placebo (p < 0.05) paralleling the in vitro observations. E2 increases NO production in endothelial cells via eNOS activation. Topical E2 application can significantly increase survival of ischemically challenged skin flaps in a rat model and may augment wound healing in other ischemic situations via activation of NO production.     

Comparison of the efficacy and safety of povidone‐iodine foam dressing (Betafoam), hydrocellular foam dressing (Allevyn), and petrolatum gauze for split‐thickness skin graft donor site dressing

We evaluated the efficacy and safety of a povidone‐iodine (PVP‐I) foam dressing (Betafoam) for donor site dressing versus a hydrocellular foam dressing (Allevyn) and petrolatum gauze. This prospective Phase 4 study was conducted between March 2016 and April 2017 at eight sites in Korea. A total of 106 consenting patients (aged ≥ 19 years, scheduled for split‐thickness skin graft) were randomised 1:1:1 to PVP‐I foam, hydrocellular, or petrolatum gauze dressings for up to 28 days after donor site collection. We assessed time to complete epithelialisation, proportion with complete epithelialisation at Day 14, and wound infection. Epithelialisation time was the shortest with PVP‐I foam dressing (12.74 ± 3.51 days) versus hydrocellular foam dressing (16.61 ± 4.45 days; P = 0.0003) and petrolatum gauze (15.06 ± 4.26 days, P = 0.0205). At Day 14, 83.87% of PVP‐I foam dressing donor sites had complete epithelialisation, versus 36.36% of hydrocellular foam dressing donor sites (P = 0.0001) and 55.88% of petrolatum gauze donor sites (P = 0.0146). There were no wound infections. Incidence rates of adverse events were comparable across groups (P = 0.1940). PVP‐I foam dressing required less time to complete epithelialisation and had a good safety profile.     

Effectiveness of collagen/oxidised regenerated cellulose/silver‐containing composite wound dressing for the treatment of medium‐depth split‐thickness skin graft donor site wounds in multi‐morbid patients: a prospective,non‐comparative,single‐centre study

Split‐thickness skin grafting (STSG) is a widely used method in reconstructive surgery, but donor site wounds (DSWs) are often slow healing and painful. This prospective study evaluated the performance of a composite wound dressing containing collagen/oxidised regenerated cellulose in the treatment of medium‐depth (0·4 mm) DSWs in 25 multi‐morbid patients with chronic leg ulcers requiring STSG. The range of patients' ages was 44–84 years (mean 71·6 years) with DSW sizes ranging between 12 and 162 cm² (mean 78 cm²). Comorbidities included anticoagulation therapy (15 patients), anaemia (11 patients), diabetes (6 patients) and methicillin‐resistant Staphylococcus aureus (MRSA) ulcer colonisation (6 patients). The first dressing change was performed after 10 days. Complete reepithelialisation was observed between the 10th and 34th day (mean 17·2, median 14 days). Postoperative medium to strong bleeding occurred in only five patients (four with anticoagulation). Wound pain levels one day after harvesting were only moderate (range 0–1·5, mean 0·5, median 0·5 on a six‐item scale). No wound infection was observed during the first dressing. The composite dressing used allowed for the fast healing of medium‐depth DSWs with minimal or no postoperative pain and bleeding in older multi‐morbid patients under anticoagulation treatment.     

Expression of CD105 and CD34 receptors controls BMP‐induced in vitro mineralization of mouse adipose‐derived stem cells but does not predict their in vivo bone‐forming potential

Adipose‐derived stem cells (ADSCs) can be excellent alternative to bone marrow derived stem cells for enhancing fracture repair since ADSCs can be isolated comparatively in large numbers from discarded lipoaspirates. However, osteogenic potential of ADSCs in vivo is very controversial. We hypothesized that adipose‐derived stem cells (ADSCs) that respond maximally to bone morphogenetic proteins (BMPs) in vitro would possess maximum bone‐forming potential. Four purified populations of mouse ADSCs: CD105⁺CD34⁺, CD105^?CD34^?, CD105⁺CD34^? and CD105^?CD34⁺ were obtained using fluorescence‐activated cell sorting (FACS) and their BMP‐responsiveness was determined in vitro. CD105⁺CD34^? population showed the strongest response to BMPs in terms of robust increase in mineralization. Expression of CD105 correlated with high BMP‐responsive phenotype and larger cell size while expression of CD34 correlated with low BMP‐responsive phenotype and smaller cell size. CD105⁺CD34^? population displayed higher gene expression of Alk1 or Alk6 receptors in comparison with other populations. However, CD105⁺CD34^? ADSCs failed to induce ectopic bone formation in vivo after they were transplanted into syngeneic mice, indicating that in vitro BMP‐responsiveness is not a good indicator to predict in vivo bone forming potential of ADSCs. Therefore greater precautions should be executed during selection of competent ADSCs for bone repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:625–632, 2015.

     

Efficacy of extended‐release tolterodine for the treatment of neurogenic detrusor overactivity and/or low‐compliance bladder

Objective: To evaluate the efficacy of extended‐release (ER) tolterodine 4 mg/day for the treatment of neurogenic detrusor overactivity (NDO) and/or low‐compliance bladder by assessing urodynamic parameters. Methods: Forty‐six patients (25 male, 21 female; mean age 57.6 ± 20.7 years) with NDO (n = 39) and/or low‐compliance bladder (n = 7) were included in this 12‐week single‐arm study. Twenty‐one patients (46%) were on clean intermittent catheterization and other patients could void on their own. A video urodynamic study was performed before and at 3 months after treatment. Changes in Overactive Bladder Symptom Score (OABSS), International Consultation on Incontinence Questionnaire–Short Form (ICIQ‐SF), and King's Health Questionnaire (KHQ) as well as changes in number of voids, amount of each void, and number of leaks in 24 h according to the 3‐day voiding diary were also evaluated before treatment and at weeks 4 and 12 after treatment. Results: Bladder capacity at first sensation and maximum cystometric capacity increased significantly, by an average of 36.8 mL (P = 0.0402) and 82.3 mL (P < 0.0001), respectively. Maximum cystometric capacity increased by more than 50 mL in 19 patients (49%) following treatment. Detrusor overactivity disappeared in three of 32 patients (9%), bladder capacity at first involuntary contraction increased significantly (P = 0.0009), and amplitude of detrusor overactivity decreased significantly (P = 0.0025). In patients with low‐compliance bladder, bladder compliance increased significantly (P = 0.0156). Overactive bladder symptom score, International Consultation on Incontinence Questionnaire–Short Form score, number of voids (per 24 h and night‐time), number of urgency episodes in 24 h, number and amount of leaks in 24 h, and amount of mean and maximum voided volumes all decreased significantly after treatment. Conclusion: Tolterodine is effective and well tolerated for the treatment of NDO and/or low‐compliance bladder in patients with neurogenic bladder.     

Attenuated Salmonella typhimurium prevents the establishment of unresectable hepatic metastases and improves survival in a murine model

Purpose

The authors investigated the utility of attenuated Salmonella typhimurium for preventing the establishment of hepatic metastases in a murine model.

Methods

A single, oral 10⁸ cfu dose of attenuated S typhimurium was given 8 days before the establishment of a model of unresectable hepatic metastases. Animals were assessed for hepatic tumor number and volume, hepatic lymphocyte population analysis, and survival.

Results

Pretreatment with Salmonella provided a 10-fold reduction in hepatic tumor burden compared with saline-treated controls. The antitumor effect is associated with markedly elevated natural killer (NK), CD8+ and CD4+ hepatic lymphocytes. Pretreatment with Salmonella provided a 90-day survival rate of 30%, whereas control animals were dead by 30 days. All long-term survivors were devoid of hepatic tumor.

Conclusions

Attenuated S typhimurium effectively prevents the establishment of hepatic metastases in a murine model, providing a clear survival benefit. Thus, it may represent a novel form of in vivo immunotherapy for the prevention of hepatic metastases for patients with locally advanced colorectal cancer.     

Hyaluronic acid dressing (Healoderm) in the treatment of diabetic foot ulcer: A prospective,randomized, placebo‐controlled,single‐center study

Fast and complete healing of a diabetic foot ulcer (DFU) is challenging due to the hostile wound healing environment of the diabetic patients. As a part of a multimodal treatment approach, advanced dressing material using hyaluronic acid (HA) has been found to be effective. However, previous studies have used HA with additional biologics, which interferes in determining the true clinical effect of HA in DFU. To examine the sole effectiveness of HA in DFU treatment, a prospective, randomized, placebo‐controlled, single‐center study was conducted using an HA dressing without additional substances. Thus, 34 patients who met the inclusion criteria were randomized into two groups (the study group: HA dressing material; the control group: conventional dressing material). During the 12‐week study period, complete ulcer healing rate was evaluated as a primary endpoint. Additionally, healing velocity and the mean duration for achieving a 50% ulcer size reduction was compared between the two groups as a secondary endpoint. At the end of the study, the study group presented a significantly higher complete healing rate as compared to that in the control group [84.6% (11/13), 41.6% (5/12), respectively, P = 0.041]. Additionally, faster ulcer healing velocity and shorter mean duration for achieving a 50% ulcer size reduction were observed in the study group (P = 0.022 and 0.004, respectively). The Kaplan‐Meier survival analysis for the median time for 50% ulcer healing rate also showed a significantly shorter duration in the study group (21 days vs. 39 days, P = 0.0127). Finally, there were no adverse events related to the dressing materials used in the study. As a major component of the extracellular matrix, this study supports the safety and efficacy of a pure HA dressing without additional substances in treating DFU.     

局部应用氨甲环酸不放置引流对降低全膝置换隐性失血的疗效

目的探讨局部应用氨甲环酸、不放置引流对降低人工全膝关节置换(TKA)后隐性失血的疗效。方法选择60例行单侧TKA患者,随机分两组,A组(30例):关节腔内应用氨甲环酸,不放置引流;B组(30例):不应用氨甲环酸,放置引流。比较两组患者术中及术后失血量、隐性失血量、输液量、输血量、患肢周径变化等指标,术前和术后3 h检查D-二聚体和相关凝血指标,术后连续复查血常规。结果术中失血量两组比较差异无统计学意义(P0.05),术后失血量和隐性失血量两组比较差异均有统计学意义(P0.05)。两组术中输液量比较差异无统计学意(P0.05),术后24 h输液量两组比较差异有统计学意义(P0.05)。术后输血量A组(480 ml±140 ml)少于B组(908 ml±248 ml)(P0.05)。术后血红蛋白和红细胞压积B组明显低于A组(P0.05)。术后3 h的D-二聚体A组为(0.92±0.45)mg/L,B组为(1.22±0.67)mg/L,均明显高于术前的(0.36±0.12)mg/L和(0.35±0.14)mg/L(P0.05),且B组高于A组(P0.05)。术后凝血指标两组比较差异无统计学意义(P0.05)。两组患肢周径变化差异均无统计学意义(P0.05)。结论TKA中局部应用氨甲环酸、不放置引流明显减少了术后隐性失血量,同时减少了输血量和输血率,且不影响凝血功能。