Extracorporeal shock waves improve angiogenesis after full thickness burn

Objective

Extensive wounds of burn patients remain a challenge due to wound infection and subsequent septicemia. We wondered whether extracorporeal shock wave application (ESWA) accelerates the healing process. The aim of the study was to analyze microcirculation, angiogenesis and leukocyte endothelium interaction after burns by using ESWA with two types of low intensity.

Methods

Full-thickness burns were inflicted to the ears of hairless mice (n = 51; area: 1.3 mm²). The mice were randomized into five groups: (A) low-energy shock waves after burn injury (0.04 mJ/mm²); (B) very low-energy shock waves after burn injury (0.015 mJ/mm²); (C) mice received burns but no ESWA (control group); (D) mice without burn were exposed to low-energy shock waves; (E) mice without burns and with no shock wave application. Intravital fluorescent microscopy was used to assess microcirculatory parameters, angiogenesis and leukocyte behavior. ESWA was performed on day 1, 3 and 7 (500 shoots, 1 Hz). Values were obtained straight after and on days 1, 3, 7 and 12 post burn.

Results

Group A showed accelerated angiogenesis (non-perfused area at day 12: 5.3% vs. 9.1% (group B) and 12.6% (group C), p = 0.005). Both shock wave groups showed improved blood flow after burn compared to group C. Shock waves significantly increased the number of rolling leukocytes compared to the non-ESWA-treated animals (group D: 210.8% vs. group E: 83.3%, p = 0.017 on day 7 and 172.3 vs. 90.9%, p = 0.01 on day 12).

Conclusion

Shock waves have a positive effect on several parameters of wound healing after burns, especially with regard to angiogenesis and leukocyte behaviour. In both ESWA groups, angiogenesis and blood flow outmatched the control group. Within the ESWA groups the higher intensity (0.04 mJ/mm²) showed better results than the lower intensity group. Moreover, shock waves increased the number of rolling and sticking leukocytes as a part of an improved metabolism.     

Influence of anti-inflammatory and vasoactive drugs on microcirculation and angiogenesis after burn in mice

Objective

The treatment of burns remains a challenge. Besides the administration of physiological saline, local disinfection and symptomatic medications, no causal therapy is known to accelerate angiogenesis and wound healing.The aim of this study was to investigate the influences of dilatative and anti-inflammatory acting drugs on microcirculation, angiogenesis and leukocyte behavior, which had shown positive effects in former burn studies.

Methods

The ears of male hairless mice (n = 47) were inflicted with full thickness burns using a hot air jet. Then the affects of five intraperitoneal injections of either acetylsalicylic acid (ASA), isosorbide dinitrate, prostaglandin E1 (PGE1) or sodium chloride (each administered to one of four corresponding study groups), on microcirculation, leukocyte-endothelial interaction and angiogenesis were investigated over a 12 day period using intravital fluorescent microscopy.

Results

Angiogenesis was slightly improved by PGE1 (0.3 vs. 1.3% non-perfused area in other groups on day 12, p = 0.029). Additionally, blood flow increased and rolling leukocytes decreased compared to other groups. The ASA-group showed best functional vessel density and lowest leukocyte-adhesion. The often described posttraumatic expansion of tissue damage could not be observed in either group.

Conclusion

Prostaglandin E1 improved angiogenesis, increased the blood flow and reduced the number of rolling leukocytes. ASA had positive influences on functional vessel density, edema formation and the number of sticking leukocytes. However, it reduced the blood flow.Overall, out of all the drugs tested, prostaglandin seems to have the greatest positive impact on microcirculation and angiogenesis in burns.     

Evaluation of angiogenesis,epithelialisation and microcirculation after application of polyhexanide,chitosan and sodium chloride in rodents

The purpose of this study was to investigate the effect of polyhexanide and a new developed chitin‐based wound dressing on skin microcirculation, epithelialisation and angiogenesis. A full‐thickness dermal layer extending to the underlying cartilage was excised on the dorsal side of hairless mice (n = 27; 2·3 ± 0·3 mm²). A polyhexanide ointment, a chitosan solution and a sodium chloride group as control were analysed using intravital fluorescence microscopy. Angiogenesis, epithelialisation and microcirculatory standard parameters were measured over a time period of 20 days. The non‐perfused area is regarded as a parameter for angiogenesis and showed the following results: on days 12, 16 and 20, the sodium chloride group was significantly superior to chitosan solution (P < 0·05) and, on days 8, 12, 16 and 20, the polyhexanide group was superior to chitosan solution (P < 0·05). The epithelialisation was measured significantly faster in the polyhexanide and control group on day 8 versus chitosan solution. Whereas polyhexanide and sodium chloride were nearly completely epithelialised, treatment with chitosan solution showed still an open wound of 11% of the initial wound size. Altogether, we could demonstrate the advantageous effects of a polyhexanide ointment on microcirculation, angiogenesis and epithelialisation. Chitosan solution appears to inhibit angiogenesis and delays epithelialisation. Further studies in different models would be worthwhile to confirm these results.     

大鼠Ⅲ度烫伤后异体、自体皮混合移植局部透明质酸含量的变化

目的 探讨混合移植皮片中细胞外间质的变化。方法 采用放射免疫方法测定了大鼠Ⅲ度烫伤后混合移植(异体皮打洞嵌植自体皮片)后3、5、7、14、21、28d自体皮区和异体皮区透明质酸的含量变化。结果 移植后自体皮区和异体皮区透明质酸含量变化均是先下降后上升，但自体皮区升高早于异体皮区，在移植后第14天(嵌植第12天)即开始显升高，至28d仍显高于正常对照，而异体皮区在移植后第21天才显升高，升高幅度也无自体皮区明显。结论 在植皮同时适当外用透明质酸可能更有利于移植皮区的愈合和重建。     

Wound healing in the bone chamber 1. Neoosteogenesis during transition from the repair to the regenerative phase in the rabbit tibial cortex

The optical bone chamber technique that includes intravital microscopy is described and is then applied to measuring primary wound healing neoostogenesis in rabbit tibia cortical bone during the period when fibrovascular tissue is being replaced by regenerating osteovascular tissue. The small population of rabbits sampled allowed only tentative conclusions. The quantitative measurements, a direct from of histomorphometry, are applied to determine the consistency of observations, with the hypothesis that healing into the bone chamber slit-gap follows the pattern of primary bone regeneration established as "characteristic" by other studies. The results supported the hypothesis and showed that bony ingrowth started during the third postoperative week (W3) with a maximum linear growth rate of 85.5 micron/day for a bone front. Evidence for remodeling by W6 was also obtained. While the bone chamber environment for tissue ingrowth is artificial, it can generate quantitative data that may provide a statistically valid basis for modeling pathophysiologic processes associated with bone wound healing.     

Extracorporeal shock wave therapy suppresses the early proinflammatory immune response to a severe cutaneous burn injury*

Following severe burn injury, persistent inflammation perpetuated by surface eschar, bacterial colonisation and neutrophil proteolytic activity can impede normal healing and result in further tissue damage. Extracorporeal shock wave treatment (ESWT) has been shown in the clinical setting to promote the healing of burn and difficult‐to‐heal wounds; however, the mechanism is unclear. We investigated the role of ESWT on the early proinflammatory response using a severe, full‐thickness and highly inflammatory cutaneous burn wound in a murine model. Various wound‐healing parameters were measured and leukocyte infiltration quantitated. A panel of 188 candidate genes known to be involved in acute inflammation and wound healing was screened. We show that ESWT of burn wounds 1 hour postwounding significantly blunts polymorphonuclear neutrophil and macrophage infiltration into the wound. ESWT treatment potently attenuates both CC‐ and CXC‐chemokine expression, acute proinflammatory cytokine expression and extracellular matrix proteolytic activity at the wound margin. Given these findings and the clinical success of ESWT, we speculate that ESWT may be a potential therapeutic modality to treat severe wounds wherein excessive inflammatory responses involving increased levels of inflammatory cells, proinflammatory cytokines and proteases may become self‐resolving allowing wound healing to progresses by way of normal physiological repair processes.     

Extracorporeal shock wave therapy role in the treatment of burn patients. A systematic literature review

IntroductionExtracorporeal shock wave therapy (ESWT), first described in the eighties for the treatment of urolithiasis, has also been applied in other fields such as orthopaedics and chronic wound care. Recently it has also been used in the treatment of burns and its sequelae since several studies suggest it could be an important tool in the conservative management of these conditions. The aim of this article is to review the literature for published evidence on the use of ESWT for the treatment of acute burn patients and its sequelae and to elaborate a brief report on the current state of the matter.Material and methodsWe carried on a search on PUBMED database and Cochrane database with the following terms: (‘burns’ [title/abstract] OR ‘burn’ [title/abstract]) AND “shock wave” ([title/abstract]). For an optimal reporting of the studies found we followed the PRISMA statement.ResultsThis search found 34 articles from which only 15 were actually related to the use of ESWT in burn patients. From these 15 articles, 7 involved the use of ESWT in the treatment of acute burns, 6 related to its application in post-burn scars, 1 in the treatment of heterotopic ossification and 1 was about the use of ESWT in skin-graft donor site. Except for the latter, all of them were carefully reviewed.ConclusionScientific evidence on the use of ESWT for the treatment of burn patients is weak due to the paucity of studies and their low quality. However, ESWT seems to be a promising tool in this field and therefore more high-quality trials should be conducted.     

Efficiency of small-volume resuscitation in restoration of disturbed skeletal muscle microcirculation after soft-tissue trauma and haemorrhagic shock

Background and aims Despite advances in primary care, trauma in conjunction with shock remains the leading cause for morbidity and mortality of young adults in western countries. Herein, we report on the efficiency of small-volume resuscitation to improve compromised perfusion of traumatised skeletal muscle tissue in shock.Methods In pentobarbital anaesthetised, mechanically ventilated rats, closed soft-tissue trauma of the right hind limb was induced, followed by induction of haemorrhagic shock [mean arterial blood pressure (MAP) 40 mmHg for 1 h]. For resuscitation, animals received saline (four-times the shed blood volume/20 min), 10% hydroxyethyl starch (HES) 200/0.5 (equal to shed blood volume/5 min) or 7.2% sodium chloride/6% hydroxyethyl starch 200/0.5 (HyperHES; 10% of shed blood volume/2 min). At 2 h of resuscitation, traumatised skeletal muscle tissue was analysed by in vivo microscopy. Non-resuscitated animals served as shock controls.Results Despite incomplete restoration of systemic blood pressure, HyperHES was superior to saline, but not to HES, with respect to amelioration of nutritive perfusion. Inflammatory cell response within the traumatised skeletal muscle tissue escaped from the anti-adhesive properties of HyperHES when applied for resuscitation from hypovolaemic shock, and did not differ from values in HES-treated and saline-treated animals.Conclusion Resuscitation with HyperHES is as effective as HES in improving capillary perfusion in traumatised skeletal muscle during haemorrhagic shock. However, because values of functional capillary density in the HyperHES-treated and HES-treated animals were still markedly below those reported in traumatised skeletal muscle of normovolaemic animals, further tools are needed to enhance efficiency in treatment of local skeletal muscle tissue injury during haemorrhagic shock.The paper was presented at the International Symposium on Significance of Musculo-Skeletal Soft Tissue on Pre-Operative Planning, Surgery and Healing, 13–14 February 2003, Berlin, Germany     

Membrane cell grafts , fresh and frozen, to cover full thickness wounds in athymic nude mice

Biobrane, an adherent, flexible temporary wound dressing was incubated with cultured human keratinocytes. The cells adhered quickly, forming ”membrane-cell-grafts” (MCG). Some of the grafts were frozen and, after thawing, viability was verified with an XTT colorimetric assay. MCGs, fresh and cryopreserved, were transplanted on full thickness wounds created on athymic nude mice and resulted in a differentiated epithelium of human phenotype. For further investigation immunohistochemistry, immunofluorescence and electronmicroscopy were performed. Conventional cultured epidermal grafts (CEG) and wounds without cell grafts served as control. Compared with CEG-grafted sites a reduced wound contraction was noticed and complete remodelling of the basement membrane zone was found. The effectiveness of the easy, uncomplicated production, cryopreservation and use, as well as the short culture period could lead to a new approach in the treatment of burns and chronic wounds. Received: 12 February 1998 / Accepted: 2 February 1999     

Ultrastructural comparison of medial collateral ligament repair after single or multiple applications of GaAlAs laser in rats

BACKGROUND AND OBJECTIVES: To examine single versus multiple applications of a gallium aluminum arsenide (GaAlAs) laser on the ultrastructural morphology of surgically injured medial collateral ligaments (MCLs) in rats. STUDY DESIGN/MATERIALS AND METHODS: Sixteen rats were studied with 12 receiving right MCL transection and 4 receiving sham injury. Group 1 (n = 4) received one session of laser (31.6 J/cm(2)) immediately after injury. Group 2 (n = 4) received 9 doses of transcutaneous laser (3.5 J/cm(2)). The controls (Group 3, n = 4) received one session of placebo laser, while the sham Group 4 (n = 4) received no treatment. Ultrastructural analyses were done with electron microscopy at 3 weeks. RESULTS: The mass-averaged diameters of collagen fibril in the core and periphery of MCLs treated with multiple laser were larger than the control and those with single laser treatment (P < 0.05). However, the sham injured group had larger fibrils than all other groups (P < 0.05). CONCLUSIONS: The repairing MCLs had smaller collagen fibrils than the sham injured ligaments. Multiple laser treatments enhanced the collagen growth in the repairing MCLs at 3 weeks after injury, which are superior to a single treatment with similar dosage.     

Acute morphological changes in canine kidneys after exposure to extracorporeal shock waves

Summary The acute effects of extracorporeal shock waves on renal morphology were studied by light and electron microscopy in 14 dogs. One kidney received an average clinical number of exposures, the nonexposed, contralateral kidney serving as control. The original Dornier HM-3 generator was used in 3 animals, the modified version in 11. Intravascular radiographic contrast medium was administered in five animals. Damage was observed in all exposed kidneys, none in the contralateral control kidney. The effects were characterized by renal and perirenal hemorrhage and edema, parenchymal hemorrhagic foci with tissue destruction, often extending from cortex to medulla. In the nearby regions there was endothelial cell damage in arteries, veins and glomerular capillaries. Breaks in the wall of these vessels were detected with platelet plug formations and thrombi. In glomeruli, breaks of Bowman's capsule and epithelial cell damage with loss of foot processes were observed. A wide range of tubular cell damage was demonstrated, ranging from vacuolization to complete necrosis. Tubular lumina were filled with red cells, indicating renal origin of hematuria. The tissue damage was less pronounced in kidneys exposed to the modified lithotriptor than to the original. No difference in the quantity or quality of damage was detected whether radiographic contrast medium was administered or not.     

微管失稳剂oncoprotein18/stathmin基因在深Ⅱ度烧伤愈合前创面及增生性瘢痕中的表达

目的观察深Ⅱ度烧伤创面愈合前及愈合后增生性瘢痕组织中微管失稳剂oncoprotein18/stathmin(Op18)基因的表达. 方法提取临床深Ⅱ度烧伤愈合前与愈合后不同时期增生性瘢痕中的总RNA,以GADPH基因表达为内参照,采用逆转录酶-多聚酶链式反应(RT-PCR)方法,半定量检测上述基因表达水平. 结果与正常组织比较,伤口愈合前Op18基因表达显著降低(P<0.05),愈合后表达迅速增高(P<0.05),在4～32个月增生性瘢痕中Op18基因稳定高表达. 结论在伤后早期Op18基因的表达水平降低与细胞增殖、组织修复的需要相适应,在增生性瘢痕组织中通过Op18基因高表达来抑制瘢痕增生.     

Cultured keratinocytes suspended in fibrin glue to cover full thickness wounds on athymic nude mice: comparison of two brands of fibrin glue

 To circumvent well-known drawbacks inherent in cultured epidermal sheet grafts, a new keratinocyte delivery system was experimently investigated. Human subconfluent keratinocytes were labeled with fluorescence cell linker and cultured in two brands of fibrin glue (Tissucol^R and Beriplast^R) in vitro for 5 days. Keratinocyte fibrin glue suspensions were grafted onto full thickness wounds in athymic nude mice for macroscopic and microscopic investigation. Keratinocytes survived in both fibrin glues in vitro for at least 5 days. When grafted onto wounds, cells maintained a high proliferation potential and at 5 days formed small masses or well-organised cell clusters within the fibrin network. After 7 days new epithelia enlarged and became thicker with high keratinization and occasional downward projections. At 10 days, wounds in both fibrin groups were totally epitheliazied but only partially in the control group. Anti-involucrin and anti-laminin immunostainings indicated well-differentiated new epithelium derived from human keratinocytes and early reorganised basement membrane. The fibrin glues seem not only to deliver highly proliferative keratinocytes easily and effectively, but also provide an optimal milieu for their migration, proliferation and differentiation. Received: 7 February 1997 / Accepted: 7 May 1997     

Evaluation of healing outcomes combining a novel polymer formulation with autologous skin cell suspension to treat deep partial and full thickness wounds in a porcine model: a pilot study

Autologous skin cell suspensions (ASCS) can treat burns of varying depths with the advantage of reduced donor site wound burden. The current standard primary dressing for ASCS is a nonabsorbant, non-adherent, perforated film (control) which has limited conformability over heterogeneous wound beds and allows for run-off of the ASCS. To address these concerns, a novel spray-on polymer formulation was tested as a potential primary dressing in porcine deep partial thickness (DPT) and full thickness (FT) wounds. It was hypothesized that the polymer would perform as well as control dressing when evaluating wound healing and scarring.DPT or FT wounds were treated with either a spray-on poly(lactic-co-glycolic acid) (PLGA) and poly(lactide-co-caprolactone) (PLCL) formulation or control ASCS dressings. Throughout the experimental time course (to day 50), we found no significant differences between polymer and control wounds in % re-epithelialization, graft-loss, epidermal or dermal thickness, or % dermal cellularity in either model. Pigmentation, erythema, elasticity, and trans-epidermal water loss (TEWL), were not significantly altered between the treatment groups, but differences between healing wounds/scars and un-injured skin were observed. No cytotoxic effect was observed in ASCS incubated with the PLGA and PLCL polymers.These data suggest that the novel spray-on polymer is a viable option as a primary dressing, with improved ease of application and conformation to irregular wounds. Polymer formulation and application technique should be a subject of future research.     

体外冲击波修复重建骨组织结构治疗股骨头缺血性坏死的临床应用

目的:探讨体外冲击波疗法促进修复重建骨组织结构治疗股骨头缺血性坏死的应用价值。方法:选择2006年3月至2008年3月CR、MRI诊断的股骨头缺血性坏死36例(42髋)应用体外冲击波治疗,男29例,女7例;年龄21～66岁,平均42.9岁;病程6个月～3年,平均1.3年。Ficat分期为Ⅰ、Ⅱ、Ⅲ期的患者分别为8、25、9髋,能量密度为0.18～0.25mJ/mm2。方法以X线透视股骨头坏死区在体表投影为中心选取1～2个冲击点为定位聚焦,每个冲击点每次冲击400～500次,每次间隔7d,共治疗3～5次。治疗前及治疗3、12个月后髋关节行X线、CT或MR检查及髋关节Harris评分,观察冲击波修复重建股骨头骨组织结构及改善髋关节功能方面的疗效。结果:42髋股骨头缺血性坏死经冲击波治疗,X线、CT或MR复查,35髋取得了良好的效果,其中治愈6髋,显效13髋,有效16髋,无效7髋。经随访1年后观察无复发,无股骨头进一步塌陷现象。治疗后3个月Ⅰ、Ⅱ、Ⅲ期髋关节Harris评分总均分分别为(90.1±5.4)、(81.2±4.5)、(66.9±5.6)分;治疗后12个月Ⅰ、Ⅱ、Ⅲ期髋关节Harris评分总均分分别为(91.6±5.4)、(91.1±5.5)、(79.0±6.1)分。均较治疗前总均分提高。结论:体外冲击波可促进修复重建骨组织结构,提高髋关节Harris评分及改善关节功能,是一种疗效明显、无创伤、并发症少、简便易行的治疗股骨头缺血性坏死的方法。