BIOCORE JOURNALS

Introduction:

Procedures for epidermal skin transplantation provide less than optimal cosmetic and functional results and cannot ensure that all skin layers are replaced^[1]. Whole (i.e. full-thickness) skin grafts that aim to produce a favourable cosmetic and functional result cannot be transplanted over large areas^[2] due to the fact that there is a limit on the size of donor area which can be used. Using cultured skin it is now possible to incorporate skin appendages in transplants, such as sweat glands and hair cells, which are extremely important for ensuring a good skin quality. Despite this, there is still no laboratory-cultured whole skin substitute that can be used on a routine basis for skin operations^[3,4]. Clinical observations have revealed that a whole skin biopsy removal using biopsy punches with a biopsy diameter of 1 - 2mm results in minimal or even no scarring. Based on this observation, a new method for intraoperative whole skin transplantation was designed in which 1 - 2mm whole skin islets recovered from biopsy punches were to be used for the purposes of an intraoperative, autologous whole skin transplantation. In a collaboration with the University of Lübeck and the Emergency Hospital Berlin, a prototype was designed for the sampling of multiple whole skin punch biopsies, each with sizes within the range of just millimetres. Using this new procedure a 5 x 5 cm sized array was designed, equipped with 1mm or 2mm biopsy punches, that was to be used for removing whole skin islets. After sampling, the whole skin islets can then be introduced into a substitute dermal matrix, after which the matrix can then be transplanted onto the new area scheduled for repair. In preliminary pilot experiments, compression and tension tests were carried out for the removal of biopsies from porcine skin using 3 mm punch diameters, and for the removal of biopsies from human cadaver skin using 2mm punch diameters. This showed that although a rotary motion significantly minimised the force required to take a successful biopsy, there was still a considerable technical challenge posed by the incorporation of a simultaneous rotary mechanism, particularly when implemented in a stamp device containing numerous individual punches. Since there have been no studies on the resection forces occurring when taking a biopsy, the focus of this paper was to compare the resection forces of 1mm and 2mm biopsy punches when employing a simple forward motion, which alone would greatly facilitate the technical realisation of a new method for carrying out whole skin transplantations.

Materials and Methods:

The forces required to penetrate the dermis and epidermis of human skin were investigated using 1 and 2mm biopsy punches. The experiments were carried out at the Laboratory of Biomechanics at the University Hospital Schleswig-Holstein using a Zwick material testing machine and its proprietary software TextXpert II (Zwick GmbH & Co. KG, Ulm, Germany). In the first test, a 100 N force sensor (load cell type Z6FD1/10 kg Hottinger Baldwin Messtechnik GmbH, Darmstadt, Germany), and in the second test, a 2000 N force sensor (load cell type U2A, 2 kN, Hottinger Baldwin Messtechnik GmbH, Darmstadt, Germany) were used to detect the forces. For the purposes of penetrating the skin, biopsy punches supplied by the company KAI of 1mm and 2mm in diameter were used (standard biopsy punches, Kai Industries & Co. Ltd., PFM Medical AG, Cologne, Germany). Human cadaver skin was used as the model tissue in the experiments carried out, which was provided by the Institute of Anatomy, University of Lübeck. These were seven frozen pieces of skin which had been obtained from the thigh of a male donor aged 79. The body parts used were examined under the auspices of the “Burial Act of Schleswig-Holstein, enacted 04/02/2005, Section II, Paragraph 9 (autopsy, anatomical)”.
The skin supporting device consisted of a base plate measuring 120 x 148 mm with 25 mm high walls as well as a perforated plate (Fig: 1).

Fig 1: Resection force test using the Zwick material testing machine.

A silicone heating mat (100 x 100 mm, 12 V, 10 W, rs-components GmbH, Mörfelden-Walldorf, Germany) was used to heat the skin in the experiments to a constant temperature of 30 ± 2 ° C. Before the test, skin was secured between the aluminium plate and the perforated plate to prevent it from moving when the tests were being conducted and to simulate and equally distribute the physiological stretching. For the first part of the experiment, 100 units of 1mm and 100 units of 2mm standard biopsy punches were used, where each consisted of a resection cylinder and a plastic overbody (Fig: 2).

Fig 2: Tensile strength test using the Zwick material testing machinemachine

The experimental setup of the first part of the experiment is shown in (Figures 3 and 4). Biopsies were taken in alternating fashion with 1mm and 2mm cylinders per hole so that for each piece of skin the biopsy punches were used 25 times with 1mm and 25 times with the 2mm punches. This procedure was carried out in the same manner with a total of four pieces of skin, so that a total measurement sample of 100 measurements with 1mm punches, and 100 measurements with 2mm punches was created (Fig: 5). In the first part of the experiment the resection forces were measured using a 100 Newton load cell with only the resection force being measured from a biopsy punch. In order to prevent incorrect values arising due to wear of the biopsy punches, a new biopsy punch was used for each measurement (i.e. 100 punches were used).

Fig 3: Punch adaptermachine
3a) 100 N force sensor with power connector 3b) Thorn attachment for the punch 3c) Skin supporting device 3d) XY table 3e) Power supply for the heating mat 3f) Control module for the material testing machine

      
Fig 4: Experimental set-up with a clamped biopsy punch            Fig 5:

For the second part of the experiment two biopsy stamps were constructed which contained either 100 x 1mm or 100 x 2mm cylinders without their plastic overbodies (Fig: 6). In these tests a 2000 N load cell was used to measure the resection forces occurring when 100 punches simultaneously penetrated the skin. It was assumed that with different punch lengths in a stamp there would be a difference in resection force compared to the stamp with equally long punches. (Fig: 7) shows the stamp with identically long punches and (Fig: 8) shows the stamp with different punch lengths. The experimental setup of the second part of the experiment is shown in (Fig: 9). Here, the resection forces of 100 x 1mm punches and 100 x 2mm punches were examined which simultaneously penetrated the skin. The stamp measurements for each skin piece were performed on three areas and on two pieces of skin per donor, meaning that a total of six measurements were made per stamp (and indeed per donor).

    
Fig 6: Implementation stamp                    Fig 7: Overall view of the prototypes

     
Fig 8: Stamp with 100 punches of different length           Fig 9: Experimental setup for the second part of the experiment
9a) 2000 N force sensor with power supply 9b) Aluminium base plate with adapter 9c) Punching stamp with 100 punches 9d) Skin layer construction with human skin

Fig 10: Resection curve for a biopsy punch
10 a) Start of measurement 10 b) Gradient 10 c) Resection the first fibre bundle 10 d) Gradient 10 e) Maximum force (or force required for rupture) 10 f) End of measurement

Results:

Figure 10 shows an example of a typical resection force curve for a 1mm biopsy punch as it penetrates the skin. The diagram reveals how the force changes in relation to the depth of penetration. In a total 8 of the 100 measurements with 1mm punches and 17 of the 100 measurements with 2mm punches, there was no cut or penetration of the skin when applying the combined forward and rotary motion (Tab: 1).

Tab 1: Number of punches that did not penetrate the skin

From the maximum forces the arithmetic mean was calculated for the individual pieces of skin, with the measured values being depicted in Table 2 below. Although all pieces of skin originated from the same donor, the resection forces measured with the four skin pieces used in the first part of the experiment varied substantially. In addition, forces measured within a single piece of skin also varied greatly.

Tab 2: Mean maximal forces per skin piece and punch

Tables 3 and 4 show the averaged and rounded-up resection forces of the 1mm and 2mm biopsy punches with the four different areas of skin. Both lower and higher resection forces are distributed over the pieces of skin, and no general organised pattern could be ascertained. The resection forces materialising with the 2mm punches were approximately twice as high as those for the 1mm punches (Table 5).

Tab 3: Measured resection force of the 1 mm punches on skin piece 4


Tab 4: Mean measured resection forces for the 1 mm punches (averaged) with the four skin pieces


Tab 5: Mean measured resection forces for the 2 mm punches (averaged) with the four skin pieces

The mean resection forces for 1mm and 2mm punches could be determined for all measuring points on the four pieces of skin. With the 1mm biopsy punch a mean resection force of 11 N arose from all 100 punches with a standard deviation of 2 N. With the 2mm biopsy punch on the other hand, the mean resection force from all 100 punches was 18 N with a standard deviation of 3 N. In addition to the tests for determining resection forces, it was also important to determine how many whole skin islets remained after resection and retraction of the punches from the skin, i.e. those that had not been completely removed from the skin. With the 2mm punches all whole skin islets were retracted from the skin by the biopsy punches. With the 1mm punches, 17 of the 100 did not cut through the skin at all, and of the remaining 83 that were cut, only 47 of these skin cylinders were successfully extracted (56.62%).

In the second part of the experiment it was assumed that the mean measured resection force for the 1mm punch from the first part of the experiment would be 100 times higher when using the specially constructed 100 punch biopsy stamp.
Of the in all 12 attempts at measurement (three measurements on four pieces of skin), resection of the skin only occurred in 7 of these cases. Table 6 shows the mean resection forces measured in the second part of the experiment for the two stamps having the same and different punch lengths. In the first part of the experiment employing single punches, a mean resection force of 11 N was determined for the 1mm punch. By employing a 100 punch stamp, as used in the second part of the experiment, a resection force of 1,100 N was expected (i.e. 100 x 11 N = 1100 N). With the stamp equipped with the same punches lengths, the mean resection force measured was surprisingly low at 639 N, i.e. only 58% of the expected 1100 N. When using the stamp with different punch lengths, an even lower mean maximum force of 478 Newton was measured, i.e. 44% of the calculated maximum force from the individual measurements.

Tab 6: Mean maximum forces and standard deviations for both stamps

Discussion:

Due to the three dimensional structure of skin, its biomechanics are extremely complex and follow a non-linear stress-strain relationship that is non-homogeneous, incompressible, anisotropic (not having the same properties in all directions), and subject to a certain prestretching and stretching force^[5,6]. To protect against external mechanical influences, the skin is also viscoelastic^[7]. These properties can be attributed to the complex network of collagen, elastin fibres and proteoglycans that are localised in the dermis^[8]. When the skin is pierced, Langer observed that the prick wound assumes an oval shape, since the skin pulls apart along what he termed "fissure" lines. Once the skin is placed under tension, the collagen-containing fibres tighten parallel to these lines^[9]. In 1933 Jochims conducted experiments to determine the mechanical resistance of skin in children. By creating a fold of skin through the pushing together of two plates placed on the skin, he measured the total force and the retraction force. The results of his measurements were that the retraction force only reflected the elastic resistances occurring, whereas the total force reflected both the elastic and inelastic (viscous) resistances^[10]. Although the elastin and collagen fibres are linearly elastic, the stress-strain curve of the skin for one-sided traction is non-linear, since the structure of the skin is not homogeneous^[11]. In 1965 Zink investigated the stretching of human skin using cadaver skin strips with a constant strain rates from a non-stretched state all the way to rupturing of the skin. More than 2,000 tests revealed a highly characteristic stress-strain relationship. Zink divided this into four phases. In the first phase, the unstretched collagen fibres are first unpleated, with the force required for this being small. During the second phase, more collagen fibres become unpleated, during which the stretching obeys Hooke's law. This stretching creates an increase in force. In the third phase, all fibre bundles are stretched and a few start to tear. In the fourth phase, a further stretching of the tissue results in a complete rupture of the collagen fibres and the tissue then starts to separate^[12]. Shergold and Fleck as well as Bischoff et al. used silicone rubber as a substitute model in some of their experiments, as this shows similar physical properties to human skin^[13,14]. However, due to its heterogeneous structure and its complexity, the design of the skin is far more complex than that of silicone rubber, and even results from animal experiments can only be extrapolated to human skin with major reservations. In our own preliminary porcine skin experiments, higher resection forces were found than was the case for human cadaver skin. Brett et al. confirmed that the force to penetrate human skin is lower than is the case with porcine skin^[15]. However, despite the use of tempered human cadaver skin, the results of this work need to be looked on with caution since after thawing of the skin an outflow of liquid was observed. Wenzel et al. found that the elasticity of the skin was related to the water content, so that the elasticity might have actually increased in the experiments described there^[16]. Although the skin pieces originated from the same donor, the resection forces between the four pieces of skin varied as an indication that the skin material was of a highly heterogeneous nature.

Through a combined forward and rotational movement of the biopsy punch, a mean resection force of just 5.6 N was measured for the 2mm biopsy punch in an earlier study. A forward rotational motion of the punch therefore results in a substantial reduction in the forces required for resection. Given that a simple forward motion showed an average resection force of 18 N, the additional rotation resulted in a reduction of the force required for resection to just 31%. Due to the fact that the resection forces were lower with simple forward movement and when using a 1mm (compared to 2mm) biopsy punch, a combined forward-rotational movement with a 1-mm-punch would lead to a further reduction of force during penetration of the skin.

Conclusion:

The aim of the work was to investigate resection forces when carrying out 1mm and 2mm biopsies from tempered human cadaver skin. In the first part of the experiment, a mean resection force of 11 N was measured for the 1mm biopsy punch, while 18 N was measured for the 2mm punch with forward movement alone. As expected, the measured resection forces when using the 2mm punch were on average higher than was the case with the 1mm punch. However, the resection forces tended to vary substantially even within skin pieces from the same donor. Based on the results of the first part of the experiment, 2 stamps consisting of 100 punches with a diameter of 1mm were constructed for the second part of the experiment. For this purpose, two types of stamps were made, where one featured identical punch lengths and the other had punches of different length. An average resection force of 639 N was determined for the stamp with equally long punches, whilst this figure was just 487 N for the stamp fitted with different punch lengths. In summary it can be concluded that very high resection forces were required to penetrate human skin with a experimental set-up involving forward punch motion alone. The authors consider a combined forward-rotational movement to be advantageous over a feed motion alone regarding the forces required to penetrate human skin with biopsy punches.

References :

1. Meek CP. Successful microdermagrafting using the Meek-Wall microdermatome. The American Journal of Surgery 1958, 96(4): 557-558
2. Billingham RE, Medawar PB. The technique of free skin grafting in mammals. Journal of Experimental Biology 1951, 28(3): 385-402
3. Chua AWC, Khoo YC, Tan BK et al. Skin tissue engineering advances in severe burns: review and therapeutic applications. Burns & Trauma 2016, 4(1): 3
4. Huh MI, An SH, Kim HG, Song YJ et al. Rapid expansion and auto-grafting efficiency of porcine full skin expanded by a skin bioreactor ex vivo. Tissue Engineering and Regenerative Medicine 2016, 13(1): 31-38
5. Griffin M, Premakumar Y, Seifalian A, Butler P, Szarko M. Biomechanical characterization of human soft tissues using indentation and tensile testing. Journal of Visualized Experiments 2016: 118
6. Bischoff JE, Arruda EM, Grosh K. Finite element modeling of human skin using an isotropic, nonlinear elastic constitutive model. Journal of Biomechanics 2000, 33(6): 645-652
7. Hendriks FM, Brokken DV, Van Eemeren JTWM, Oomens CWJ, et al. A numerical‐experimental method to characterize the non‐linear mechanical behaviour of human skin. Skin Research and Technology 2003, 9(3): 274-283
8. Silver FH, Freeman JW, DeVore D. Viscoelastic properties of human skin and processed dermis. Skin Research and Technology 2001, 7(1): 18-23
9. Langer K. On the anatomy and physiology of the skin: I. The cleavability of the cutis. British Journal of Plastic Surgery 1978, 31(1): 3-8
10. Jochims J. Untersuchungen des mechanischen Verhaltens der Hautgewebe (Cutis+ Subcutis) mit einer neuen Methode. Zeitschrift für Kinderheilkunde 1934, 56(1): 81-97
11. Daly CH, Odland GF. Age-related changes in the mechanical properties of human skin. Journal of Investigative Dermatology 1979, 73(1): 84-87
12. Zink P. Methoden zur Bestimmung der mechanischen Eigenschaften der menschlichen Leichenhaut. Deutsche Zeitschrift für die gesamte gerichtliche Medizin 1965, 56(6): 349-370
13. Shergold OA, Fleck NA. Experimental investigation into the deep penetration of soft solids by sharp and blunt punches, with application to the piercing of skin. Journal of Biomechanical Engineering 2005, 127(5): 838-848
14. Bischoff JE, Arruda EM, Grosh K. Finite element modeling of human skin using an isotropic, nonlinear elastic constitutive model. Journal of Biomechanics 2000, 33(6): 645-652
15. Brett PN, Parker TJ, Harrison AS et al. Simulation of resistance forces acting on surgical needles. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine 1997, 211(4): 335-347