Hydroxyethyl starch solution for extracorporeal tissue perfusion

Abstract

In the field of free flap transfer in reconstructive surgery, the trans- or replanted tissue always undergoes cell damage during ischemia to a more or less strong extent. In previous studies we already showed that conserving muscle transplants by means of extracorporeal perfusion over a period of 6 hours by using a crystalloid solution for perfusion. However, we observed significant edema formation. In this study we aimed at reducing the edema formation by using an iso-oncotic colloid as perfusion solution. This way we wanted to evaluate a possible new application of hydroxyl-ethyl starch in an extracorporeal setup to exploit potential benefits of the colloid.

Examined parameters include the muscles’ functionality with external field stimulation, histological examination and edema formation. Perfused muscles showed a statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, as perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. Substituting the electrolyte perfusion solution with a colloidal one shows the tendency to reduce the edema formation however without statistical significance.

Keywords

Extracorporeal perfusion hydroxyethyl starch free flap ischemia transplantation

1 Introduction

In the field of organ transplantation, replantation of amputated extremities and free flap transfer in reconstructive surgery, the trans- or replanted tissue always undergoes cell damage during ischemia to a more or less strong extent [3, 22]. This phase of ischemia is the time when the tissue is disconnected from the blood circulation, consequently suffering from insufficient oxygen and nutrient supply as well as metabolic product removal. Muscle tissue is very sensitive towards ischemia. To reduce the need of autologous tissue transplantations, methods of tissue engineering seem promising in this context, but are not clinically available options yet [12, 13].

In previous studies we already showed that conserving muscle transplants from pigs by means of extracorporeal perfusion over a period of 1 hour is superior to conventional storage protocols [6–8 , 28].Only recently we were able to extend our results from previous studies over an extracorporeal perfusion period of 6 hours by using a crystalloid solution for perfusion [27]. External field stimulation and contraction force measurements were introduced as new methods besides immunohistochemical staining of Annexin V to assess the viability of the muscle and thus, to evaluate the effectiveness of extracorporeal perfusion in preventing ischemia-related cell damage [27]. Despite promising results, we observed significant edema formation of the muscle with an average weight gain of 99.9% (±22.5%) after 6 hours of perfusion with an isotonic crystalloid solution [27]. The use of an isotonic crystalloid solution for fluid replacement in daily clinical routine is known to cause a diffusion of the infused solution into the entire extracellular space which is distributed 20% intravascular and 80% interstitial [27]. In contrast to this, iso-oncotic colloids are known to remain mainly intravasal (60% –90%) [5], depending on the mean molecular weight, the C2/C6 hydroxyethylation ratio and the degree of substitution [16, 29]. Depending on the molecular structure and concentration, colloids can lead to an increase but also to a decrease of capillary perfusion [19].

Because an expanding edema impairs the diffusion and thus affects tissue oxygenation [10], in this study we aimed at reducing the edema formation by using an iso-oncotic colloid (i.e. a hydroxyethyl starch (HES) based solution) as perfusion solution. Apart from the perfusion solution the experimental set-up was used as previously described to ensure comparison with previous results [27]: One porcine rectus abdominis muscle was continuously perfused with the iso-oncotic colloid, whereas the contralateral muscle, which was used as control, only received a stationary flush of the same colloid. At this point it should, however, be mentioned that the risk assessment committee of the European Medicines Agency restricted the application of hydroxyethyl starches for the treatment of acute hypovolemia caused by acute blood loss in June 2013 [5]. Though still discussed controversially [4 , 31], an increased risk for kidney injury and a higher mortality was observed when critically ill patients were treated with hydroxyethyl starch [5]. In this study, we used an isolated muscle to investigate the effects of a colloid-containing solution on the quality of muscle tissue conservation. Possible harmful effects on interior organs are thus obsolete but potential effects on muscle tissue directly can be examined. This way we wanted to evaluate a possible new application of HES in an extracorporeal setup to exploit potential benefits of the colloid with eliminating possible risks.

2 Methods

2.1 Animals and surgical protocol

Animals were treated like in our former study [27]. Hereby, one rectus abdominis muscle from male mature pigs (n = 5; Erzeugergemeinschaft Franken Schwaben, Tierische Veredelung, Wertingen-Geratshofen, Germany) was harvested with its vascular pedicle (inferior epigastric artery and veins) for perfusion, the other one was used as control. Preoperatively, the pigs were sedated for 30 minutes with an intramuscular injection of atropine (Atropinsulfat^®, Braun, Germany; 0.044 mg/kg) and azaperone (Stresnil^®, Janssen, Germany; 4 mg/kg). Anaesthesia was performed with an intravenous injection of ketamine (Ketanest® 10%, Ceva, Düsseldorf, Deutschland; 15 mg/kg) and pentobarbital (Narcoren^®, Hallbergmoos, Deutschland; diluted 1:2; 20–40 mg/kg). Orotracheal intubation (6.5–7.0 Ch) was performed after topical anaesthesia of the larynx (lidocaine, Xylocain^®; AstraZeneca, London, UK) under laryngoscopic control. No muscle relaxant was used in order not to disturb the contraction studies. For ventilation, intermittent positive pressure ventilation with weight-adapted respiration volume was done. Anaesthesia was maintained by inhalation of a gas mixture of 1.5–2% isoflurane (Forene^®, Abbott GmbH & Co. KG, Wiesbaden, Germany) with air/oxygen. During operation, the animals received weight-adapted crystalloids (Jonosteril^®, Fresenius Kabi, Bad Homburg, Germany).

For muscle harvest, a skin incision was made after disinfection starting at the xyphoideal processus continuing to the symphysis. The ventral rectus sheath was opened and the muscle with its caudal vascular pedicle was harvested. Hereby, the muscle was dissected from its lowest point up to the third or fourth intersection.

All experiments were approved by the Government of Mittelfranken, Germany No. 65- 2532.2-1/10 and the animal care committee of the Friedrich-Alexander-University of Erlangen–Nürnberg. All experiments were carried out in accordance with the relevant guidelines and regulations.

2.2 Extracorporeal perfusion and external field stimulation

The perfusion protocol, respectively the treatment of the control group of the rectus abdominis muscle flaps, was performed like in a previous study [27], except that as perfusate a colloid was used (Volulyte^® 6%, mean molecular weight: 130.000 Da, molar substitution: 0,38–0,45, C2/C6 hydroxyethylation ratio: 9:1, Fresenius Kabi, Bad Homburg, Germany). Hereby, both rectus abdominis muscles were harvested –one for perfusion experiments, the other acted as control. While the control flap received only a single shot of 20 ml heparinized colloid via its arterial branch to remove residual blood like in clinical daily routine, the artery and vein of the perfused flap were cannulated and actively perfused with the colloid using a pressure controlled pump (flow rate: 600 ml/h). The perfusate was supplied from a second circuit that includes an oxygenator (SAFE Micro^®, Polystan, Denmark) and received the venous drainage from the muscle. The experiments were performed over 6 hours at ambient temperature. In the perfusate, oxygen levels at the arterial and venous branch were monitored using optical sensors (Sensor type: PSt3 FTC, Transmitter: OXY - 4 - mini, Presens Precision Sensing GmbH; Regensburg; Germany). In addition, systolic pressure was monitored at the arterial branch (SIEMENS Sirecust 961^®, Siemens AG, Munich, Germany). Both muscles were fixed with clamps in separated Perspex bioreactors, each filled with 15 l of electrolyte solution. These bioreactors contained isometric force gauges (Alluristrademark FMI-220B2, Alluris GmbH, Freiburg, Germany) attached to one upper clamp. In front of and behind the flaps, silver plate electrodes (fine silver 999, dimensions: 120 mm×50 mm) were placed with a distance of 6 cm between the electrodes, each connected to an electrical stimulator (Myotronictrademark Stimulator, Myotronic UG, Heidelberg, Germany). Hereby, contactless external field stimulation was performed and the exerted force of the muscles was recorded in order to assess viability throughout the experiment. Stimulation setup was as follows: The frequency of stimuli was set to 100 Hz, generating a fused tetanus of muscle fibers, while the operation mode was adjusted to monophasic square waved current. The square waves were seen every 0.01 s with a pulse duration of 1 ms. Over 10 s bouts of tetani were applied. Pretension of the muscles was set to 0.25 N and adjusted whenever necessary. Stimulation was performed every 15 minutes, whereas stimulation pulses were triggered three times in a row. Muscles were stimulated with an initial stimulation voltage of 0.166 V·cm^–1.

2.3 Histology

Muscle samples were taken at time point 0 and at the end of the experiment after 6 hours. To preserve validity of the contraction measurements no further samples were taken during the ongoing experiment. Biopsies were fixed in 4% buffered formalin at ambient temperature before embedding in paraffin. Haematoxylin and Eosin (H&E) staining was performed as described in the protocol of the Leica Autostainer XL (Leica Biosystems Nussloch GmbH, Nussloch, Germany).

Immunohistochemistry (IHC) was performed using antibodies against Annexin V. As described previously, nuclei positive for Annexin V labelling are an indicator for progressed cell damage in a later phase of apoptosis [27]. The slides (2-3 μm) were pretreated with a sodium citrate buffer (pH 6.0). Then, the slides were flushed with a tris-buffer, followed by the addition of a blocking solution (Reagent 1) 5 min before overnight incubation with antibodies (dilution 1:500) against Annexin V. The Fast-Red-reaction was added for 15–20 min (room temperature) just after PostBlock (Reagent 2) for 30 min and AP-Polymer (Reagent 3) for another 30 min was added. For nuclei detection, a counterstaining of the section with Haematoxylin was performed before mounting.

For analysis all slides were captured using a digital slide scanner (Panoramic-Midi; and Panoramic Flash 250, 3D-Histech AG, Budapest, Hungary). Regarding IHC, the slides were analyzed independently by two specialists to achieve double-blinded quality controls. Hereby, five high power fields (HPFs) (magnification of 200, studies area: 600×400 μm) were randomly distributed over each slide and documented for quality management. Only nuclei located at the inner border of the plasma membrane of skeletal muscle cells were recorded. Nuclei outside the plasma membrane or close to capillaries were not counted as they may be indicative of non-muscle origin, e.g. blood cells or endothelium. With a 400× magnification, the counted nuclei in the HPFs were differentiated in positive and negative ones.

2.4 Edema formation

To quantify edema formation, especially regarding the perfused muscle flaps, the flaps’ weights were documented before and after the stimulation experiments (i.e. time point 0 and 6 hours).

2.5 Statistical analysis

For statistical analysis, a two-sided paired student’s test was performed, where a p-value of <0.05 was considered significant. The analyses were done using SPSS for Windows version 22 (SPSS Inc., Chicago, USA) software.

2.6 Perfusion solution

To flush the flaps (control, 20 ml once), respectively for continuous perfusion (600 ml/h), heparinized (5,000 I.E./liter) Volulyte^® 6% (Fresenius Kabi Deutschland) was used.

3 Results

3.1 Preservation of muscular function

The conservation protocol has an impact on muscular function. The recorded forces of the muscles decline over the time period of six hours when suffering from ischemia as well as when treated with continuous perfusion, but the decline can be reduced in the latter case. After initialising the stimulation at time point 0 (Fig. 1) the muscles exert a maximum force in the first second after stimulation. After the transient maximum, force declines rapidly despite the ongoing stimulation and transitions into a steady state like force with a small force decline after three to four seconds after intialized stimulation.

Noticable is the rapid decline in the muscles exerted force induced by EFS. In the presented exemplary case the muscle generates a maximum force between 5.9 N and 7.3 N, while after one hour of ischemia, this decreased to a value between 0.5 N and 0.6 N. At the end of the measurement period of ten seconds, the initial force ranges between 0.9 N and 1.4 N and after one hour between 0.1 N and 0.2 N.

Both parameters, maximum force as well as steady force, have been used to determine the success of continuous perfusion to preserve muscular function. In order to relate force decrements from different muscles of different length and cross-section to initial force as a relative measure between muscle flaps, the following normalization procedure proved to be useful: recorded forces at different points of time are normalized both with reference to the voltage used for stimulation as well as to the value of effective force.

The resulting effective maximum force at different points of time is shown in Fig. 2.

Although a decline in effective maximum force can be observed for both interventions, continuous perfusion and singular flush without further treatment, a difference in the muscles’ tetanic force can be observed. Within the control group, suffering from ischemia after receiving a flush of Volulyte^®, the effective force diminishes after 2 hours of ischemia to 3.5% of the highest achievable effective force. This level is maintained during ongoing ischemia with an increasing number of muscles failing to exert force after electrical field stimulation (EFS). After 3 hours ex vivo two muscles did not react to EFS, another failed after 5 hours, and after 6 hours of ischemia, all muscles were no longer able to exert a force higher than the threshold of 0.5 N. In contrast, continuous perfusion with the HES solution resulted in a nearly stable effective force of all muscles between 3 and 6 hours ex vivo without any muscles failing. The effective maximum force of all time points are summarized in Table 1.

During the first two hours ex vivo no statistical significance between the perfused and control, group can be observed. However after 2 hours, the difference in effective maximum force becomes statistically significant.

The resulting effective steady force at different points of time is shown in Fig. 3.

The effective steady force of all muscles declines as well during the time course of the experiment. Muscles in the control group already exert a significantly lower effective force after one hour of ischemia, 20.1% of the initial value, and are only to exert 3.1% of the initial effective force after 2 hours. After 3 hours ex vivo, muscles start to fail to exert any steady force as it was the case with two muscles. After 5 hours only one muscle reacted to EFS and after 6 hours of ischemia, all muscles were no longer able to exert a force. The initial effective steady forces of the group undergoing continuous perfusion do not coincide with the highest recorded effictive forces. While this is also the case with the control group, as some muscles reach the highest effective force after fifteen minutes of the experiment, some muscles exert their highest effective force after one or two hours of perfusion. After 3 hours of perfusion though, the effective steady force declines slowly, reaching 18.0% at the end of the recorded time. The effective steady force of all time points are summarized in Table 2.

In accordance with the effective maximum force, the difference in effective steady force becomes statistically significant after two hours into the experiment.

3.2 Histology

In addition to the effective force decline, a difference in Annexin V positive nuclei between 6 hours of ischemia or perfusion can be observed. Figure 4 shows representative images of immunohistochemically stained slides of muscle samples stained for H&E (Fig. 4a–c) and against Annexin V (Fig. 4d–f).

First it has to be mentioned that even immediately after disconnection from the natural blood circulation, before undergoing further treatment Annexin V positive nuclei can be found, resulting in a ratio of positive nuclei of 27.7% (±9.3%) in the group before ischemia and 23.0% (±9.2%) in the group before undergoing perfusion can be found, without a statistical significant difference between both groups (p = 0.25). After six hours ex vivo significant changes occur. In muscles undergoing six hours of ischemia, the ratio of positive nuclei rises by 19.4% (±10.1%; p = 0.002) to a total of 47.0% (±4.0%), while in perfused muscles, this value is augmented only by 6.7% (±4.9%) to an average of 30.0% (±6.6%; p = 0.13) (see Fig. 5).

To summarize, continuous perfusion prevents a statistically significant rise in Annexin V positive nuclei that occurs in muscles suffering from ischemia.

3.3 Oxygen consumption

The oxygen consumption of the perfused muscles is determined continuously during active perfusion. By comparing the oxygen partial pressure (pO₂) of the perfusate on arterial and venous side, an oxygen consumption of the muscle tissue can be determined (Fig. 6). The pO₂ on arterial side is maintained at 100% ambient air saturation by a secondary reoxygenation circuit containing a micro-porous polypropylene membrane.

Initially the perfused tissue consumes 61.9% (±18.7%) of the supplied oxygen. During further perfusion, oxygen consumption is reduced over the first hour of the experiment: 51.5% (±10.2%), but reaches a stable value between 44.8% (±9.6%) and 50.3% (±6.2%) after 2 hours of perfusion. The nonperfused muscle is considered to be suffering from a complete lack of oxygen, as has been shown in previous publications [8].

3.4 Perfusion pressure

Arterial pressure has been recorded over the time course of the experiment. After an initial maximum arterial pressure at an average of 121 mmHg (±33 mmHg), the pressure decreases slowly and reaches a stable plateau between 93.5 mmHg (±48 mmHg) and 96.3 mmHg (±42 mmHg) after 3 hours of perfusion.

3.5 Edema formation

The main drawback of a saline perfusion solution is the high probability for the formation of an interstitial and intracellular edema, even at low perfusion pressures. The intention is that by increasing colloidal osmotic pressure the edema formation should be reduced. The formation of an edema can be seen by evaluation of the muscle’s change in weight after perfusion. After six hours of extracorporeal perfusion, the average weight gain is 84.0% (±25.1%). Without perfusion, there is almost no change in weight after 6 hours (8.1%;±1.7%).

4 Discussion

This work has been performed to show the improvement of muscle preservation using a colloidal solution in comparison to a standard electrolyte solution which has been evaluated earlier. The results using the saline solution have already been published including a detailed methodology evaluation [27]. The same analysis has been used in this work including the preservation of muscle contractility, histological analysis, tissue oxygen consumption and perfusion pressure which are also used to look at the muscle conservation improvement using a hydroxyethyl starch solution.

Using a colloidal solution like HES for extracorporeal perfusion the formation of an interstitial and intracellular edema is reduced to a certain degree comparing to perfusion with a simple saline solution, which was used earlier. Using the identical perfusion setup with the saline solution a weight gain of about 100% (99.9% ±22.5%) was observed [27]. Substituting the crystalloid solution with one containing a colloid a weak trend can be observed as the mean weight gain is 14% lower, without statistical significance (p = 0.44). Introducing a colloid into the fluid system increases intravasal oncotic pressure so that the diffusion of water into the interstitium is decreased. The formation of an edema is not averted completely. Reducing the overall perfusion pressure averts the formation of an edema but this can only be achieved by decreasing the flow rate of the perfusate. A lower velocity and consequently a higher residence time of the perfusate in turn have a negative impact on the supply with oxygen at the end of the vascular system as well as on the removal of undesired metabolites [15]. Increased concentrations of HES as well as chemically modified version of HES, i.e. longer chain lengths and higher degrees of substitution, increase the colloidal osmotic pressure but also the viscosity and finally, the overall perfusion pressure. The latter leads to steep pressure drops, which have to be avoided by all means to prevent damage to the vascular system. The use of other osmotically active substances like albumin may be considered, but albumin is not ideally suitable for a medical product intended for single usage in this setting due to high costs [2 , 20]. Beyond all that HES is known to improve circulation in macro- and microvessels in vivo, depending on the molecular structure and concentration [17 , 24].

Next to the effect on edema formation, the use of HES has an influence on other results as well. The muscles’ ability to react to EFS is somewhat improved, especially when used in a continuously perfused setting as the average effective force after six hours of artificial circulation with HES is higher than with the saline solution. At the end of the trial of six hours, HES perfused muscles are still able to exert 11.7% of the original peak value, while those receiving the saline solution decline to a level of 2.6% [27]. The curve progression in both cases is pretty similar with a high loss in effective force during the first two hours of perfusion followed by a slow but steady decline in effective force during the rest of the experiment. The same can be observed with muscles treated only by a single flush of the respective perfusate. In both ischemia cases though, flushed with a saline solution as well as HES, all muscles fail to react to EFS after six hours of ischemia (Table 3).

A small effect on the ratio of Annexin V positive nuclei can be observed using HES as perfusate and flush to remove intravasal blood. Muscles suffering from ischemia with HES in the circulatory system show an average increase in positive nuclei of 19.4% (±10.1%), while the same setting with saline solution results in a rise of 24.4% (±6.5%). These cannot be confirmed with a continuous perfusion as both changes in Annexin V ratios over 6 hours are in the same range: 6.7% (±4.9%) when using HES and 6.3% (±4.8%) when using saline solution.

Using HES for hemodilution is known to improve tissue oxygen pressure and flow properties of blood compared to physiologic saline solutions [9]. No effect on the muscles oxygen uptake can be seen in this study when comparing colloid and crystalloid solution. Both perfusion groups extract up to 50% of the administered oxygen that is solved in the perfusate (between 44.8% and 50.3% using HES and between 46.1% and 48.1% using saline solution). Again, not all of the oxygen is extracted from the perfusate, half still resides in the liquid. This means in this perfusion setting, ambient air saturated perfusate with a flow rate of 10 ml/min, an adequate amount of oxygen can be supplied for the conserved muscle. No additional oxygen carriers like hemoglobin or artificial equivalents are necessary to ensure a sufficient supply with oxygen. The nonperfused muscle is considered to be suffering from a complete lack of oxygen, as has been shown in previous publications [8].

Although the results are promising there is still room for improvements. First the edema problem has to be resolved. In addition to apparent harmful effects, diffusion of molecules into and out of the tissue is hindered due to water intake. Second, no additional nutrients are included in the perfusate, its main objectives are limited to oxygen supply, carbon dioxide removal and the balancing electrolyte levels. Introducing nutrients may help maintaining normal metabolism that can improve functionality and viability [14 , 30]. Third, a change in temperature may have additional beneficial effects on the conservation of muscle tissue. Hypothermia slows down cellular metabolism and thus postpones the accumulation of toxic metabolites. Furthermore, oxygen and nutrient demand is reduced so that less complex solutions are satisfactory [1]. The other approach is to keep the tissue under normothermic conditions to maintain cellular metabolism to utilize intracellular repair and adaptation mechanisms for conservation. The drawback is the demand of an complex perfusion solution to ensure a steady supply of all major nutrients [30].

Next to optimization of the aforementioned parameters, the experimental setup itself can be improved. The clinical use of HES solutions has been widely discussed. The European Medicines Agency restricted the application of hydroxyethyl starches [28]. HES based solutions are contraindicated in the case of patients suffering from sepsis, burns, impaired renal function, intracranial or cerebral bleeding, clinically ill patients, severe coagulation disorder, severe liver dysfunction, hyperhydration and dehydration. HES based solutions are only to be administered when the treatment of hypovolemia from severe blood loss with crystalloid solutions is not sufficient [5]. Basically, the intended use of these colloids has been included in these recommendations. Though being discussed controversially, we could show that with these colloids the occurance and extent of an edema formation can be decreased to a certain degree. Next to the perfusate, the perfusion method can be modified as well. At the moment, the fluid is pumped in a circulatory system. That means the fluid is collected in a storage tank and after reoxygenation and degassing of carbon dioxide reintroduced into the system. This can only be sustained for a limited amount of time, as next to reoxygenation, other ingredients have to be resupplied as well as electrolytes and potential nutrients. A simplification of the delivery system might be considered. Instead of a circulatory perfusion system a one-way system may suffice. This would make regeneration elements unnecessary and would make the system easy to use in clinical application. As a consequence, the perfusate has to be designed as a disposable. Without the need of artificial oxygen carriers and sustaining the osmotic pressure through HES instead of albumin, the perfusate can be kept affordable and economically usable as a disposable.

Footnotes

Acknowledgments

This study was funded by the ‘ELAN Fonds für Forschung und Lehre’.

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