Manuel Held

In vivo biocompatibility and biodegradation of a novel thin and mechanically stable collagen scaffold.

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable, and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabeled for CD68 and stained by hematoxylin-eosin and Masson-Goldners trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long-term vascularization and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption, and replacement by autologous tissue. The in vivo biocompatibility and degradation behavior encourage for further evaluation of CCC in surgical applications and regenerative medicine.

Development of an animal frostbite injury model using the Goettingen-Minipig

BACKGROUND A standardized reproducible animal model is desirable to gain a better understanding in the pathophysiology of frostbite injury and to develop novel therapies. Little has been studied about frostbite injury models. The depth and extent were not always being controllable and consistent. Therefore, we developed a frostbite injury model using the Göttingen Minipigs. METHODS 42 frostbite injuries were inflicted on the abdomen of seven minipigs using an aluminium bar (300g, circular with a radius of 1cm) frozen with liquid nitrogen to -196°C. The bar was applied for 1, 3, 6, 12, 30 and 60s. Five hours after injury biopsies were performed and evaluated by the presence of denatured collagen, blood vessels, follicular epithelial cells and mesenchymal cells. RESULTS A large spectrum of frostbite injuries was obtained ranging from superficial, superficial partial, deep partial to full thickness. The depth of injury in the sets of frostbites was highly consistent (Pearson correlation ranged from r=0.89 to 0.94). CONCLUSION We described a simple, inexpensive and safe swine frostbite model. The accurate reproducibility of the depth of the frostbite injuries allows researchers to accurately plan frostbite studies.

A quantitative analysis of microcirculation in sore-prone pressure areas on conventional and pressure relief hospital mattresses using laser Doppler flowmetry and tissue spectrophotometry

BACKGROUND Pressure ulcers are associated with severe impairment for the patients and high economic load. With this study we wanted to gain more insight to the skin perfusion dynamics due to external loading. Furthermore, we evaluated the effect of different types of pressure relief mattresses. METHODS A total of 25 healthy volunteers were enrolled in the study. Perfusion dynamics of the sacral and the heel area were assessed using the O2C-device, which combines a laser light, to determine blood flow, and white light to determine the relative amount of hemoglobin. Three mattresses were evaluated compared to a hard surface: a standard hospital foam mattress bed, a visco-elastic foam mattress, and an air-fluidized bed. RESULTS In the heel area, only the air-fluidized bed was able to maintain the blood circulation (mean blood flow of 13.6 ± 6 versus 3.9 ± 3 AU and mean relative amount of hemoglobin of 44.0 ± 14 versus 32.7 ± 12 AU.) In the sacral area, all used mattresses revealed an improvement of blood circulation compared to the hard surface. CONCLUSION The results of this study form a more precise pattern of perfusion changes due to external loading on various pressure relief mattresses. This knowledge may reduce the incidence of pressure ulcers and may be an influencing factor in pressure relief mattress selection.

Improvement of Skin Quality Using a New Collagen Scaffold in Acute Burns and Reconstructive Surgery: An In Vivo Evaluation of Split Skin Graft Transplantation in a Rat Model

Introduction Split‐thickness skin grafting is often associated with poor skin quality. In this context, a new collagen cell carrier (CCC), as an underlayment in split skin graft (SSG) transplantation for covering tissue defects in rats, has been evaluated as an improving agent. Methods Twenty‐eight full‐thickness wounds were covered with SSGs, applying the CCC as an underlayment in 14 rats and using SSG transplantation alone in control group. Postgraft skin areas were assessed using an instrument that measures mechanical properties of skin. Three parameters were considered for skin elasticity analysis: total skin deviation (R0), gross elasticity (R2) and viscoelasticity (R8). Measurements were performed every 10 days for 80 days after grafting. Biopsies were taken subsequently for histologic evaluation. Results The results demonstrated significantly superior elasticity values in CCC‐supplemented SSGs in terms of gross elasticity and viscoelasticity (R2/R8) starting from day 60 after grafting to the end of the measuring period. There was no histologic evidence of inflammation, adverse host tissue reaction, or scar tissue formation. Conclusion Split skin grafting is associated with poor dermal quality, but CCC may offer unique opportunities in complex wound management in terms of skin graft quality improvement.

Alteration of biomechanical properties of burned skin

BACKGROUND The prevalence of burns in the general population is high. Despite new research findings, skin burns and its resulting tissue damage are still not entirely understood. In particular, little is known about the depth-dependent alteration of skin biomechanical properties of these wounds. METHODS Thirty-six burn wounds with six different depths were generated on the abdomen of six Göttingen minipigs. The alteration of skin biomechanical properties was evaluated objectively after 15 and 360 min using a Cutometer device. Biopsies for histological evaluation were taken and the depth of burn was correlated with biomechanical properties. RESULTS Firmness of skin (R0), overall elasticity (R8) and calculated elasticity (Ue) demonstrated a continuous decrease with an increasing depth of burn 15 min after wound generation. Gross elasticity (R2), net elasticity (R5) and amount of elasticity of the whole curve (R7), however, showed an increase of values with increasing depth of injury. A further decrease of elasticity was demonstrated 360 min after wound generation. CONCLUSION The alteration of skin biomechanical properties is a function of damaged tissue structures. The presented results demonstrate a depth-dependent decrease of principal elastic parameters with an increasing depth of burn and the results indicate progressive tissue damage over the time.

The Effect of Repetitive Intraperitoneal Anesthesia by Application of Fentanyl-Medetomidine and Midazolam in Laboratory Rats

ABSTRACT Background: Literature reviews show numerous options for anesthesia in the small laboratory animals. Many methods are associated with complications, such as high technical effort, difficult monitoring, respiratory and cardiovascular depression, and prolonged sedation. In the present study, we report first time results after repeated use of an intraperitoneal combined anesthesia with a high tolerability. Methods: Three hundred and seventy-four anesthesias were performed on 38 adult male lewis rats (280–460g). Each animal was anesthetized repeatedly over a period of three months, using an intraperitoneal combination of Fentanyl-Medetomidine and Midazolam (FMM). The time required for the animals to lose ear pinch response and the ability to perform a righting and pedal withdrawal reflex was measured. For evaluation of the clinical state, a four-point vitality scale was developed. The anesthesia was antagonized with Naloxone, Flumazenil, and Atipamezole (s.c.). Results: The animals lost all three reflex responses within 5 (± 2.4) min of injection. Without antagonism of anesthesia, the ear pinch response returned on average within 125 (± 21.5) min. After antagonism of anesthesia, the rats needed 5 (± 2.9) min to regain all three reflex responses. No significant differences of vitality-index were measured after repeated use of FMM during the investigation period. Conclusions: A repeatable and secure anesthesia is indispensable for any experimental studies that require multiple anesthesia of a single animal. Intraperitoneal combination of FMM provides an adequate procedure to induce a well tolerable, repeatable state of anesthesia, which conforms to all the necessary requirements for laboratory rats.

Assessment of microcirculatory changes of cold contact injuries in a swine model using laser Doppler flowmetry and tissue spectrophotometry

BACKGROUND Until now, the exact pathophysiology of frostbite injuries is poorly understood. The aim of the study is to evaluate the perfusion changes of frostbite injuries to get a better understanding of the exact mechanism underlying the resulting tissue damage. Particular attention has been given to the differentiation of changes between the various injury depths. METHODS Cold contact injury ranging from superficial, superficial-partial, deep-partial to full thickness were generated using seven goettingen minipigs. The perfusion dynamics were assessed before and 3 h after the injuries were inflicted using the O2C-device, which combines a laser light, to determine blood flow, and white light to determine hemoglobin oxygenation and relative amount of hemoglobin. A total of 42 cold contact injuries were inflicted and 84 measurements were carried out. RESULTS In superficial and superficial partial injuries there was an increase in the blood flow (mean, 20 ± 2.4%, 15 ± 5.1%, respectively) and in the relative amount of hemoglobin (mean, 29 ± 9.1%, 28 ± 7.2%, respectively), whereas the hemoglobin oxygenation did not alter (mean, -0.15 ± 2.4%, -0.8 ± 3.1%, respectively). In deep partial injuries there was a decrease in the blood flow (mean range, -4 ± 2.1 to -17 ± 4.7%) and an increase in the relative amount of hemoglobin (mean range, 79 ± 17.2 to 93 ± 17.7%), whereas the hemoglobin oxygenation did not alter (mean range, -0.7 ± 3.1 to -2.9 ± 3.3%). In full thickness injuries there was a drop in the blood flow (mean range, -24 ± 7.6 to -27 ± 11.8%) and in the hemoglobin oxygenation (mean range, -16 ± 3.5 to -19 ± 2.5%) and an increase in the relative amount of hemoglobin (mean range, +126 ± 19.8 to 145 ± 10.9%). CONCLUSION The results of this study form a more precise pattern of cold contact injury perfusion changes, which can be a valuable tool to assess the degree of cold contact injury and furthermore the efficacy of novel diagnostic aids, therapeutics and treatment modalities.

Alteration of biomechanical properties of skin in acute cold contact injury

BACKGROUND The prevalence of acute cold injury has increased recently. Despite new research findings, these injuries and their resulting tissue damage are still not entirely understood. Especially, little is known about alteration of skin biomechanical properties. METHODS A total of 36 acute cold contact wounds with different depths were generated on the abdomen of six Göttingen minipigs. Alteration of biomechanical properties of skin was evaluated objectively after 15 and 360 min using a Cutometer device. Biopsies for histological evaluation were taken, and the depth of injury was correlated with biomechanical properties. RESULTS Calculated elasticity (Ue), firmness of skin (R0) and overall elasticity (R8) demonstrated a continuous decrease, whereas other parameters demonstrated an initial increase with increasing depth of injury 15 min after wound generation. All parameters showed an increase compared to healthy skin, 360 min after wound generation. Furthermore, an alteration of values over time was detected. CONCLUSION Alteration of biomechanical properties of skin is a function of damaged tissue structures. The presented results demonstrate a decrease of main elastic parameters with increasing depth of injury and indicate progressive tissue damage over time. Skin elasticity measurements are a valuable tool in acute cold contact injury depth assessment and may act as an influencing factor in management decisions.

Evaluation of Two Commonly Used Temporary Skin Dressings for the Treatment of Acute Partial-Thickness Wounds in Rats

OBJECTIVES There is a broad range of expensive temporary wound dressings that promise better results in wound management. The aim of this study was to compare two commonly used temporary dressings for the treatment of partial‐thickness wounds in a rat model. METHODS Forty‐two partial thickness wounds were created on the back of Lewis rats and treated with the dressings; control wounds remained without treatment. Wound size was determined daily by measuring the scabbed area. Three months after the wounds were created, skin elasticity was measured and a histologic evaluation was performed. RESULTS Wound appearance in the animals in the treated groups did not differ significantly. Wound closure was slower in the control group than in the dressing groups. There was no histologic evidence of inflammation and no suggestion of epidermal changes in any group. CONCLUSION Using both skin dressings, we observed satisfying results without any significant differences. Because of rising health care expenses, cost should play an essential role in the clinical application of these dressings.

A new, rapid, standardized method for harvesting split skin grafts in rodents.

Background: Harvesting split skin grafts in rodents has proven to be challenging, as the presence of panniculus carnosus tissue in rodent skin causes high flexibility and mobility of the upper skin layer. Thus, fixation is indispensable for granting precise manipulation during the grafting process. All techniques established in the literature achieve stability by dissecting a subcutaneous pocket beneath the panniculus carnosus. In this article, the authors present a method that excludes inflicting additional wounds other than that to the donor site but takes advantage of the elasticity of rat skin. Methods: A customized sturdy support plate is held laterally beside the skin area where the split skin is to be cut. The skin is mobilized, pulled over the support plate, and fixed with the nondominant hand on the bottom of the device. Using a dermatome, the split skin graft is cut on the metal plate, which provides a flat surface to permit a precise harvesting process. Results: A total of 208 split skin grafts were harvested, of which nine had to be rejected. The average time to perform a split skin graft on one animal, not considering preparation time, was 2 minutes. All of the harvested grafts were 2 × 2.5 cm (0.79 × 0.98 inch), with a mean thickness of 0.3 mm. Reharvesting could be performed on postoperative day 7. Conclusions: Using this new standardized method, grafts of equal size and thickness can be obtained rapidly. It is cheap and highly reproducible and can be carried out by a single surgeon. It provides a rapid healing process with fewer complications and immensely reduced stress for the animals.