Human preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo.

Currently, there is no adequate implant material for the correction of soft tissue defects such as after extensive deep burns, after tumor resection and in hereditary and congenital defects (e.g. Romberg's disease, Poland syndrome). The autologous transplantation of mature adipose tissue has poor results. In this study human preadipocytes of young adults were isolated and cultured. 10(6) preadipocytes were seeded onto collagen sponges with uniform 40 microm pore size and regular lamellar structure and implanted into immunodeficient mice. Collagen sponges without preadipocytes were used in the controls. Macroscopical impression, weight, thickness, histology, immunohistochemistry (scaffold structure, cellularity, penetration depth of the seeded cells) and ultrastructure were assessed after 24 h in vitro and after explantation at 3 and 8 weeks. Preadipocytes penetrated the scaffolds 24 h after seeding at a depth of 299+/-55 microm before implantation. Macroscopically after 3 and 8 weeks in vivo layers of adipose tissue accompanied by new vessels were found on all preadipocyte/collagen grafts. The control grafts appeared unchanged without vessel ingrowth. There was a significant weight loss of all grafts between 24 h in vitro and 3 weeks in vivo (p < 0.05), whereas there was only a slight weight reduction from week 3 to 8. The thickness decreased in the first 3 weeks (p < 0.05) in all grafts. The preadipocyte/collagen grafts were thinner but had a higher weight than the controls at this point in time. The histology showed adipose tissue and a rich vascularisation adherent to the scaffolds under a capsule. The control sponges contained only few cells and a capsule but no adipose tissue. Human-vimentin positive cells were found in all preadipocyte/collagen grafts but not in the controls, penetrating 1188+/-498 microm (3 weeks) and 1433+/-685 microm (8 weeks). Ultrastructural analysis showed complete in vivo differentiation of viable adipocytes in the sponge seeded with preadipocytes. Formation of extracellular matrix was more pronounced in the preadipocyte/collagen grafts. The transplantation of isolated and cultured preadipocytes within a standardised collagen matrix resulted in well-vascularised adipose-like tissue. It is assumed that a pore size greater than 40 microm is required, as preadipocytes enlarge during differentiation due to incorporation of lipids.

Inflammatory response to a porcine membrane composed of fibrous collagen and elastin as dermal substitute.

The inflammatory response to a collagen/elastin membrane was studied by measuring the expression of cytokines and function associated antigens in human macrophages. Additionally the angiogenic and inflammatory activity in the chorioallantoic membrane of the chick embryo (CAM-assay) was investigated. Macrophages cultured on the membrane expressed IL-1beta mRNA as early as after 4 hours. During prolonged culturing IL-1beta mRNA levels decreased. Messenger RNA for IL-8 was detectable over the whole culture period. The anti-inflammatory cytokine IL-10 was expressed up to one day only. Phenotypic analysis revealed a decrease in the number of chronic inflammatory 25F9 positive macrophages not migrating into the membrane but a presence of these cells together with the acute inflammatory 27E10 macrophages within the membrane whereas the anti-inflammatory subtype RM3/1 was absent. In the CAM-assay the membrane stimulated angiogenesis and induced the formation of granulation tissue. Histological analysis showed that the membrane was infiltrated with macrophages, fibroblasts and endothelial cells and locally with granulocytes. These data show that the collagen/elastin membrane causes activation of macrophages, angiogenesis and the formation of inflammatory tissue. Although these processes are essential for wound healing the type of inflammation points to a chronic process which might counteract an efficient scar formation.

Cross-linking by 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane meant to be used as a dermal substitute: effects on physical, biochemical and biological features in vitro.

Next to in vitro-cultured autogeneic keratinocytes for the restoration of epidermis, a suitable dermal matrix is a mandatory component of an artificial skin substitute for the permanent covering of full thickness skin defects. In our model a xenogeneic membrane, consisting of processed native collagen and elastin of porcine origin is meant to serve as a template for the formation of a neo-dermis. In order to improve the resistance of this matrix against enzymatical degradation, we cross-linked it by using the carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) together with N-hydroxysuccinimide. Chemical cross-linking by these agents at two different degrees (shrinkage temperatures 63 degrees C and 81 degrees C) had no relevant effect on mechanical features or water-uptake capacity. The time needed for enzymatic digestion was increased by cross-linking. Concerning growth and spreading of fibroblasts and keratinocytes on and within the structure of this membrane, we did not observe a difference between cross-linked and non-cross-linked material (shrinkage temperature 48 degrees C). We therefore expect that cross-linking by EDC is an effective means to control the degradation of the collagen/elastin membranes in vivo without a significant influence on their biocompatibility.

The chorioallantoic membrane of the chick embryo as a simple model for the study of the angiogenic and inflammatory response to biomaterials.

Angiogenesis is essential in wound healing and a common feature in chronic inflammation which is crucially involved in the biological response to biomaterials. A useful system to evaluate the angiogenic activity and the inflammatory potency of various agents is the chorioallantoic membrane (CAM) of the chick embryo. Here we examined its response to different biomaterials. Smooth materials such as PVC or the polyurethane Tecoflex either unmodified or modified by an OH- or N(CH(3))(3)(+)-end group (HEMA or MAPTAC) inhibited angiogenesis and did not induce the formation of granulation tissue. The anti-angiogenic effects of PVC, Tecoflex and its HEMA modification, however, were only seen at an early stage of development. In contrast, the MAPTAC modified Tecoflex inhibited angiogenesis over the whole time. Rough materials, e.g. filter paper or a collagen/elastin membrane, stimulated angiogenesis and induced the formation of inflammatory tissue. Histological analysis revealed that the filter material was homogeneously populated with cells consisiting mainly of macrophages, fibroblasts and endothelial cells. The collagen/elastin membrane was only partially infiltrated with cells. Among those also clusters of granulocytes were present pointing to an acute inflammatory process. These data show that the angiogenic activity and inflammatory response of biomaterials strongly depend on the chemical composition and the physical structure of the material. The CAM assay appears to be a useful tool for studying biocompatibility.

C1 inhibitor prevents capillary leakage after thermal trauma.

OBJECTIVE: In burned patients, activation of the complement and clotting systems is suggested to play an important role in the development of the capillary leak syndrome and inflammatory tissue destruction. In an animal model of thermal trauma, the possible protective effect of C1 inhibitor (C1Inh), a major control protein of both the complement and clotting systems, was investigated. DESIGN: Prospective, controlled study. SETTING: Animal model. SUBJECTS: Healthy pigs weighing 30 kg. INTERVENTIONS: Pigs were scalded for 25 secs with 75 degrees C hot water to achieve a 30% total body surface deep partial-thickness burn. The treatment group (n = 8) received C1Inh concentrate at an initial dose of 100 units/kg body weight immediately after thermal trauma, followed by three further applications every 12 hrs. Two control groups included animals that were either scalded (n = 8) or not scalded (n = 7) and treated with lactated Ringer's solution. MEASUREMENTS: Before and at various time points after trauma blood samples were analyzed for complement activation (APH50, CH50, SC5b-9, C3). Continuous monitoring of hemodynamic variables was performed and postmortem histologic examination of specimens from lung, heart, liver, kidney, stomach, duodenum, jejunum, ileum, and colon was carried out. Aseptically collected mesenteric lymph nodes were pooled and screened for bacterial translocation. For evaluation of the burn wound, biopsies from defined scalded and not scalded areas were taken daily. As a measure for edema formation, the weight of the animals was recorded every 2 hrs. RESULTS: After C1Inh treatment, which led to a significantly reduced complement activation, the clinical outcome was clearly improved, as indicated by vital signs and as demonstrated by reduced edema formation. Treated animals presented a diminished bacterial translocation. Pathologic alterations were clearly diminished in the burned skin, in shock-related organs, and in the intestines. CONCLUSION: Application of C1Inh appears to be an effective means to prevent capillary leakage and inflammatory tissue destruction after thermal trauma.

A new technique for quantitative bacterial assessment on burn wounds by modified dermabrasion.

Bacterial colonization and invasive bacterial infection is still one of the major problems in the treatment of burn victims. The standard procedures of bacterial monitoring of the burn would are i) swab-culture which is non-invasive but detects bacteria at the very surface and ii) biopsy-culture which gives a more complete view but has the disadvantage of being invasive. Therefore we developed a new technique for examination of microbial colonization of the wound surface. Dermabrasion of the upper layers of the wound was performed using a small rotating carbon-steel disc of defined roughness. The tissue samples obtained were analysed for bacterial growth in different culture media. Results were qualitatively and quantitatively compared with those of standard techniques performed in parallel. Our results show that this new technique is superior to the swab culture in identifying different bacterial species. The results can be compared with the biopsy technique, but has the advantage of being less invasive.

Combination anesthesia with ketamine and pentobarbital: a long-term porcine model.

Anesthesia of the pig poses great problems for experimental animal-based research and particularly in shock research. In this study, five mechanically ventilated domestic pigs were given long-term anesthesia with a combination of ketamine plus pentobarbital. Circulatory parameters were recorded every 2 h via an arterial catheter placed in the right common carotid artery, a Swan-Gans thermodilution catheter (7F), that was placed in the pulmonary artery of the right middle-lobe in a wedge position through the external jugular vein, and another catheter in the internal jugular vein for measuring central venous pressure. Moreover, body weight, blood gases, pH, blood cells, electrolytes and serum enzymes were measured. Further serum traits as total protein and glucose and pathological alterations in different organs were recorded. The animals were observed for a period of 96 h and then killed painlessly. It was shown that pigs can survive 96-h anesthesia with the combination of ketamine and pentobarbital. Optimum, carefully controlled anesthesia did not impair the integrity of the regulatory mechanisms of circulation.

Use of a collagen/elastin-membrane for the tissue engineering of dermis.

In an experimental model in rats, xenogeneic membranes consisting of processed native collagen and elastin were grafted to serve as a template for the formation of a neo-dermis, while in vitro-cultured autogeneic keratinocytes were applied on top of this to restore an epidermis. The process of tissue reconstruction and the digestion of the grafted membrane components were analysed by histological and immunohistochemical methods as well as electron microscopy. Approximately 3 weeks post grafting the membranes were completely vascularised and colonized by different types of cells. After 6 weeks, the collagenous fibres of the graft were mostly replaced by newly formed collagenous texture, whereas elastic membrane components were still present even after 20 weeks. In a second step, in vitro-cultured keratinocytes were applied onto the partially integrated membranes, resulting in an epithelial coverage of approximately 47% of the grafted area after 8-11 days. As early as on day 6 post application, a multilayered, partially differentiated epithelium, together with lymphocytes and Langerhans' cells, could be observed. After 10 days the formation of a basement membrane including anchoring fibrils appeared to be complete. This three-dimensional matrix structure offers a promising scaffold for a tissue engineering strategy to restore skin structure and function. Further experimental studies are in progress to test its applicability to human skin replacement.

The influence of the C1-inhibitor BERINERT and the protein-free haemodialysate ACTIHAEMYL20% on the evolution of the depth of scald burns in a porcine model.

Standardized deep partial-thickness burns were inflicted on domestic pigs by scalding 30 per cent of the skin surface for 25 s with 75 degrees C hot water. The animals (n = 18; weight 25-35 kg) were divided into three groups: I, control group (n = 6), Ringer's lactate only; II, haemodialysate group (n = 6), Ringer's lactate and a protein-free haemodialysate of calf-blood (ACTIHAEMYL20%; AH) and III, C1-inhibitor group (n = 6), Ringer's lactate and C1-inhibitor (C1-INH; BERINERT). Skin biopsies were taken at defined time points (4, 28, 52 and 76 h) and investigated histologically. Depth of burn was determined morphometrically after coloration with a modified MTT-staining on frozen sections of the skin biopsies. Fluid therapy with C1-INH decelerated significantly the progression of the burn wound in the postburn-period compared to Ringer's lactate alone. In comparison with C1-INH, the treatment with AH demonstrated a less beneficial influence on the depth of scald burns. The favourable effects of C1-INH are explained by the protection of the dermal microcirculation during the acute phase of thermal injury.

[Objective evaluation of the microcirculation in the skin with indocyanine green angiography (ICGA). A method for the clinic?]. / Objektive Beurteilung der Mikrozirkulation an der Haut mit der Indozyanin-Grün-Angiographie (ICGA). Eine Methode für die Klinik?

In some cases, assessment of the microcirculation of tissues can be very difficult. Various methods have been proposed for evaluating microcirculation, none of which has so far established itself for clinical use. Indocyanine Green Angiography (ICGA) is a suitable and objective method for quantifying microcirculation and due to the pharmacocinetic properties of this dye the prospects for clinical application of this fluorescence technique are rather promising. In an experimental animal study, postoperative changes of microcirculation were examined in a skin flap. The day of operation, ICGA showed pathophysiologic changes of the microcirculation which was corrected during the postoperative phase. This new state of haemodynamic equilibrium was objectively quantified by ICGA. The objectivity and repeatability within short time intervals speak for clinical application after adequate development and experience. The indication has to be clarified in each case. Rare incidence of negative reactions should be kept in mind.

[In vitro and in vivo studies of a temporary absorbable dressing]. / In-vitro- und in-vivo-Untersuchungen einer absorbierbaren temporären Wundabdeckung.

The major advantage of biodegradable synthetic wound dressings compared to other synthetic materials and biological tissue derivatives is avoidance of the necessity to change the dressing. The copolymer films used in this study were made of lactic and caproic acid. They pose no problems concerning immunology or transmission of diseases. Moreover, the substances released by degradation may possibly influence the process of wound healing. The material proved to be non-toxic and was used successfully as a carrier for cell culture with keratinocytes. The permeability for bacteria is determined by degradation. Evaluation of the bacteriological studies indicates secure protection against secondary contamination of the films for at least 15 days, however. Concerning application, dressing properties, wound healing and wound retraction, no disadvantages were found in comparison with the results of the competition's film Opsite. Based on these findings, the films are now used in a clinical study as a wound dressing for split-skin donor sites.

Treatment of skin defects using suspensions of in vitro cultured keratinocytes.

The in vitro cultivation of keratinocytes and their application in the form of confluent sheets to cover various kinds of skin defects involves a number of problematical steps which could be improved by using single cell suspensions instead. Therefore we developed a method to apply keratinocytes suspended as single cells in a fibrin gel. By testing the feasibility of this method in different experimental animal models we found that it facilitates cultivation as well as application of the cells, moreover, this method allows a much more flexible use of the cells, i.e. it is easier to consider the clinical condition of the patient than by the conventional method.

The immunogenicity of glycerol-preserved donor skin.

Previous clinical observations have suggested that the application of glycerol-preserved donor skin as a temporary wound dressing provokes a weaker rejection reaction than fresh, vital donor skin. Like others, we frequently observed that considerable parts of the allodermis not only remained on the wound for an extended period of time, but even became re-epithelialized in some cases. In order to quantify this effect, we applied the mixed lymphocyte culture (MLC) test in a rat model, using the two highly inbred, histoincompatible rat strains DA and Lewis as donor and recipient respectively. Using the methodology of the Euro Skin Bank, Beverwijk, The Netherlands, split thickness skin, excised from the back of the rats, was equilibrated in 98 per cent glycerol. The immunological reaction after grafting vital DA-skin, glycerolized DA-skin onto Lewis rats, and vital as well as glycerolized Lewis-skin onto Lewis rats was compared. The results of these experiments do not support the clinical observations that the glycerolization procedure results in decreased immunogenicity of donor skin.

A computerised machine for the facilitated production of intermingled skin grafts.

The introduction of intermingled allograft/autograft skin grafting in western countries has long been hampered by high costs, due to the personnel requirements to produce this special form of graft. We have solved this problem by designing and constructing a computer-controlled machine, which in one operation punches holes out of strips of allogeneic donor skin and in another cuts fitting islets out of autogenous skin and transfers them into these holes. In comparison to manual preparation, this machine not only accelerates the production of intermingled skin grafts, but it also inserts the islets with superior accuracy, which is very important for the final functional and cosmetic result.

Increased survival rates by topical treatment of burns with cerium nitrate.

In experiments on mice we were able to show that the negative effects attributed to burn toxins could almost completely be prevented by one single early treatment of the burned skin with a 0.04 M solution of cerium nitrate [Ce(NO3)3]. The survival rate was 10% for animals which were grafted with burned skin. Treatment with Ce(NO3)3 increased the survival rate to 74%. A reflection of this protective effect was the prevention of the burn-induced disturbance of the acceptor control ratio in isolated liver cell mitochondria. Repeated use of Ce(NO3)3 showed adverse effects due to an increased absorption. An effective treatment of burns with Ce(NO3)3 is without problems and can be done in any hospital.

Intermingled skin grafts with in vitro cultured keratinocytes--experiments with rats.

In vitro cultured autogenic keratinocytes were used instead of autogenic skin islets in intermingled skin grafts on rats. According to the observations made with the original method, the allogenic epidermis is being rejected. The cultured keratinocytes start to grow out in circles, eventually covering the remaining allodermis in the form of a multilayered, well-differentiated epidermis. The prospects of these findings for possible future clinical applications are discussed.

Distribution of lymphocytes in intermingled skin grafts.

Chinese intermingled skin grafts of allogenic material interspersed with small autogenic islets heal permanently with no signs of a rejection reaction. A study of the T-helper and T-suppressor cells in the region of the autogenic islets and the remaining allodermis revealed a distinctly greater frequency of these cells in the islets, with marked massing of the cells between the autoepithelium and the autodermis. Histologically it could be shown, that the Langerhans' cells grow over the allodermis together with the epithelium, although compared with the autogenic islets their number in the newly formed epidermis remained reduced.

Cytogenetic investigations of the allodermis after intermingled skin grafting.

In most instances intermingled skin transplants done according to the method developed in the PR China are not subject to a generalized rejection. During the healing process there is an integration of the allodermis instead, which eventually becomes covered by outgrowing auto-epidermal cells. To investigate the fate of allogenic cells experiments were done with intermingled skin grafts, using male Wistar rats as recipients and female DA-rats as donors. By studying karyotype analysis of cells growing out from intermingled skin biopsies the presence of viable allogenic cells was demonstrated on days 53 and 111 post-transplantation. It was concluded that the intermingled skin forms a cellular mosaic with different portions of autogenic and allogenic cells.

A model to reproduce predictable full-thickness burns in an experimental animal.

A standardized, reproducible animal model is a prerequisite to study concepts in the therapy of extensive burn injuries. The development of a new model makes it possible to produce predetermined burn injuries with a set temperature, time, contact pressure, and standard extent of tissue damage. For our studies we chose rats and exposed them to a temperature of 250 degrees C for 20 s and a contact pressure of 500 g/cm2 over various percentages of TBSA (total body surface area). The animals received shock prophylaxis for 3 days postburn and were kept under standardized conditions in a laminar airflow compartment. The temperature was kept at 32 degrees C and the relative humidity at 75 per cent. To reduce bacterial contamination, air was filtered through special bacteria-proof filters. Under these conditions we found burns of approximately 35 per cent TBSA to be sublethal resulting in 80 per cent mortality between days 5 and 7. This model permits the investigator to vary the burned skin area to any required extent for a reproducible study of different concepts of burn therapy.

Morphological changes of intermingled skin transplants on rats.

Experiments with rats demonstrate that free skin transplants between genetically distinct strains become totally rejected after no longer than 3 weeks. In intermingled skin transplants, however, the autogenic epidermis directly covers the allodermis, which results in a definitively covered wound with a mostly normal skin texture. It seems that during the first phase after the burn the allodermis is accepted as donor skin. In the second phase it becomes covered by the epidermis and surrounded by the connective tissue of the recipient. During this process all structures of ectodermal origin like skin adnexa are rejected. Since for the most part the elastic fibres in the allograft remain unchanged after a successful intermingled skin transplantation, the transplant shows elasticity similar to a mesh autograft, but it is incapable of regenerating skin adnexa. After complete healing cellular and humoral infiltrates, as in the case of immunological rejections, could no longer be detected.