Evaluation of Copper and Hydrogen Peroxide Treatments on the Biology,Biomechanics,and Cytotoxicity of Decellularized Dermal Allografts

Abstract

Decellularized tissue allografts are paving the way as an alternative to cellular tissue transplantation. Effective sterilization or decontamination of tissue allografts is paramount for the safety of the allograft; however, some of the current sterilization procedures have a detrimental effect on the tissue scaffold. The bactericidal and virucidal activity of copper (II) ions and hydrogen peroxide (H₂O₂) have been widely reported, however, their effect on the biology, biochemistry, and biocompatibility of decellularized tissue have yet to be elucidated. In this study, decellularized human dermis (dCELL human dermis) was treated with copper (II) chloride (CuCl₂) and H₂O₂; both singly and in combination, and parameters, including concentration, pH, and synergy between CuCl₂ and H₂O₂, were evaluated to identify conditions where any detrimental effects on the tissue scaffold were observed. Skin from 13 human donors was retrieved with appropriate consent and processed into dCELL human dermis. The dCELL human dermis was then treated for 3 h with 0.1 mg/L–1 g/L (w/v) CuCl₂ and 0.01–7.5% (v/v) H₂O₂ and combinations of both of these in the same concentration range. dCELL human dermis treated with solutions of 0.1 mg/L–1 g/L CuCl₂ or 0.01–7.5% H₂O₂ caused no detrimental effects on gross histology, collagen denaturation, collagen orientation, and biomechanical properties of the tissue or cytotoxicity. The highest combined concentration of CuCl₂ and H₂O₂ demonstrated an increase in ultimate tensile strength, loss of collagen type IV immunostaining at the dermal-epidermal junction, and in vitro cytotoxicity. Combinations within the range of up to 10 mg/L CuCl₂ with up to 0.5% H₂O₂ had no effect. The data identify the concentrations of CuCl₂ and H₂O₂ solutions that have no effect on the biological, biomechanical, and biochemical properties of dCELL human dermis, while retaining biocompatibility. These treatments may be suitable for use as sterilization/decontamination agents on human decellularized tissues.

Introduction

While rare, contamination of tissue allografts can result in mortality of the recipient.¹ Therefore sterilization of the allograft before transplantation is vitally important. Fresh viable allografts can only be disinfected due to the damaging effect sterilants have on graft cells.² For nonviable allografts, for example, glycerol-preserved skin, a range of sterilants can be applied depending on the tissue type. However, due to methods of preservation, dead cells can elicit some cytotoxicity and immunogenicity thereby compromising recellularization and graft incorporation.^3,4 Tissue banks have tended to use one of two sterilization processes; irradiation or chemical sterilization using ethylene oxide. Gamma irradiation is a common sterilization method, regularly used to terminally sterilize skin and bone allografts.^4,5 However, recent data suggest that application of gamma irradiation above a certain threshold dose can affect the structural integrity of these grafts and render them cytotoxic.^6–8 The use of ethylene oxide as a sterilization agent is becoming less common due to concerns over possible genotoxic effects and poor penetration of denser tissues and occupational exposure has been linked to health complications.^9–11 Peracetic acid (PAA) has been investigated as a potential alternative chemical sterilant, however, there are conflicting views over its use particularly for soft tissue allografts. PAA has been shown to be a suitable sterilant for skin allografts, having no effect on the viscoelasticity, biomechanical function, and biocompatibility of these tissues.^12,13 Conversely, PAA-induced impairment of biomechanical function and revascularization of other soft-tissue allografts, for example, ligaments, have been reported.¹⁴

Decellularized tissue allografts have been used for reconstructive surgical applications and transplantation for a number of years. The removal of cellular components from tissue allografts renders these decellularized grafts nonimmunogenic, allowing recellularization of the scaffold by the recipient's own cells.^15–17 Decellularized tissue eliminates the concern of effect the sterilization procedure has on the graft cells, however, the effect of sterilants on the extracellular matrix scaffold needs to be taken into consideration. Studies have shown that PAA has a negative effect on type IV collagen at the basement membrane of acellular blood vessels and dCELL human dermis.^18,19 Gouk et al. demonstrated that gamma irradiation of decellularized dermis reduced the elasticity, increased susceptibility to enzymatic degradation, and induced structural modifications that subsequently compromised the tensile strength of the tissue.²⁰ Thus, the need for the development of alternative sterilization methods is evident.

The sterilization activity of metal-based and other formulations, for example, cupric [copper (II)] ions or hydrogen peroxide (H₂O₂), is widely recognized. Several experimental studies have documented the antimicrobial and virucidal activity of cupric ions.^21,22 Such reagents are utilized in water sanitization processes and the healthcare industry to disinfect reusable medical equipment.^23,24 Copper-impregnated contact surfaces for example, trolleys or bed rails, have been shown to significantly reduce the bacterial bioburden compared to noncopper surfaces and reduce the rate of hospital-acquired infections.^25,26 H₂O₂ in both liquid and gaseous states is used in the medical, food, and environmental industry as a broad-spectrum biocide, including efficacy against bacterial spores.²⁷ NHS Blood and Transplant (NHSBT) incorporates H₂O₂ into wash solutions during bone allograft processing as a means to reduce pathogen contamination without compromising the osteoinductivity and biomechanical stability of the bone.^28–30 The potentiation of the bactericidal and virucidal activity of copper ions by H₂O₂ is well documented.^21,22,31,32 In addition, Solassol et al. showed that combinations of cupric ions and H₂O₂ reduced the levels of prion protein (PrP^sc) in infected brain homogenates to a greater degree than individual treatments.³³

To date, no chemical formulation containing a combination of copper (II) chloride (CuCl₂) and H₂O₂ has been used as a sterilization agent in tissue banking or tissue engineering. Development of a new tissue sterilant must first establish that the candidate sterilant itself does not have any adverse effect on the tissue. In this study, using decellularized (dCELL) human dermis developed by NHSBT,^19,34 we investigate a range of CuCl₂ and H₂O₂ concentrations and pH values, used individually and in combination, to identify those treatments that do not damage dCELL human dermis. Evaluation of the effects of CuCl₂ and H₂O₂ was determined by conventional histology and biomechanical analysis. Dermal collagen was assessed by immunohistochemistry, collagen birefringence, and hydroxyproline content. Cytotoxicity of treated dCELL dermis was evaluated by an in vitro contact assay. Our findings identify those CuCl₂ and H₂O₂ treatments/concentrations that preserve the morphology, functionality, and biocompatibility of dCELL human dermis. These treatments/concentrations can subsequently be evaluated for their potential as a tissue sterilant.

Methods

Tissue

Cryopreserved skin from 13 deceased donors (males, aged 50–83 years old) was used in this study. All donor families had given full consent for R&D use. The donor was shaved and then washed with 0.5% chlorhexidine (EcoLab) and phosphate-buffered saline (PBS; Sigma Aldrich). Strips of skin (5–6 × 30 cm) were retrieved by trained Tissue Services Donation staff from the legs and the back and sides of the torso using a dermatome (Braun) set to a nominal depth of 0.33 mm. The skin was treated with an antibiotic cocktail (Cambridge Antibiotics; Source BioScience), cryopreserved in 20% v/v glycerol (Source BioScience), and stored at −80°C until use.

Dermis decellularization

Skin was thawed, washed in PBS, and decellularized as described in Hogg et al. Briefly, the epidermis was removed by incubating skin in 1 M sodium chloride (Source BioScience) for 18 h (37°C); the dermis was then washed in PBS and incubated in hypotonic buffer (Source BioScience) for 16 h (4°C). Dermis was washed in PBS and incubated for a further 24 h (25°C) in a 0.01% (w/v) sodium dodecyl sulfate detergent buffer (Source Bioscience). After another PBS wash, dermis was transferred to a nuclease buffer (Source BioScience) containing 1 U/mL benzonase (Merck Millipore) for 3 h (37°C). The proteinase inhibitor aprotinin (10 KIU/mL) and the antibiotics penicillin–streptomycin (1% v/v) (Sigma Aldrich) were used in the incubations. After decellularization, the dCELL human dermis was washed in PBS +0.1% EDTA (VWR) and frozen at −40°C until use.

Stability of copper chloride and hydrogen peroxide solutions

Solutions of 0.1, 1, 10, and 100 mg/L and 1 g/L (w/v) CuCl₂ (Sigma Aldrich) at pH 3, 4, and 5 were prepared in distilled water and stored at 4°C. Reagent grade (30%) H₂O₂ (Sigma Aldrich) was used to prepare solutions of 0.01, 0.1, 0.5, 1.0, 3.0, and 7.5% (v/v) in distilled water at pH 3, 4, and 5 and were stored at 4°C. Solutions containing a combination of CuCl₂ and H₂O₂ were made in the concentration range of 0.1–100 mg/L CuCl₂ + 0.01–3.0% H₂O₂ and stored similarly. Combining these concentrations of CuCl₂ and H₂O₂, the pH was maintained below pH 5 and therefore there was no need for adjustment (Table 1). To establish if there was any degradation of copper or hydrogen peroxide over time (44 days), the concentration of copper (II) was determined photometrically using an AQUANAL™-plus copper (Cu) kit (Sigma Aldrich) according to the manufacturer's instructions. H₂O₂ levels were measured photometrically using a quantitative peroxidase kit (Thermo Scientific) according to the manufacturer's instructions.

Table 1.

pH of Solutions Containing 0.1–100 mg/L CuCl₂ in Combination with 0.01–7.5% H₂O₂ Over 44 Days

	pH at 24 h	pH at 44 days
0.1 mg/L CuCl₂
0.01% H₂O₂	4.4 ± 1.0	4.7 ± 0.5
0.1% H₂O₂	4.2 ± 1.0	4.5 ± 0.4
0.5% H₂O₂	4.2 ± 1.3	4.3 ± 0.6
1% H₂O₂	4.0 ± 1.0	4.2 ± 0.6
3% H₂O₂	3.7 ± 0.8	ND
7.5% H₂O₂	3.5 ± 0. 7	ND
1 mg/L CuCl₂
0.01% H₂O₂	4.9 ± 0.3	4.3 ± 0.6
0.1% H₂O₂	4.5 ± 1.4	4.4 ± 0.5
0.5% H₂O₂	4.2 ± 1.7	4.2 ± 1.2
1% H₂O₂	4.2 ± 1.9	4.2 ± 1.5
3% H₂O₂	3.6 ± 1.2	ND
7.5% H₂O₂	3.5 ± 1.1	ND
10 mg/L CuCl₂
0.01% H₂O₂	4.2 ± 0.2	4.2 ± 0.1
0.1% H₂O₂	4.0 ± 0.6	4.1 ± 0.4
0.5% H₂O₂	3.8 ± 0.7	3.9 ± 0.6
1% H₂O₂	3.7 ± 0.9	3.7 ± 0.7
3% H₂O₂	3.5 ± 1.0	ND
7.5% H₂O₂	3.3 ± 1.1	ND
100 mg/L CuCl₂
0.01% H₂O₂	4.3 ± 0.2	4.4 ± 0.1
0.1% H₂O₂	4.5 ± 0.8	4.5 ± 0.5
0.5% H₂O₂	3.9 ± 0.2	4.0 ± 0.5
1% H₂O₂	3.7 ± 0.2	3.8 ± 0.5
3% H₂O₂	3.4 ± 0.6	ND
7.5% H₂O₂	3.1 ± 0.5	ND

At 44 days, due to a reduction in the measured concentrations of CuCl₂ when combined with 3% and 7.5% H₂O₂, the pH of these solutions were not determined (ND). Values are means ± 95 CI.

Copper chloride and hydrogen peroxide treatment

dCELL human dermis was treated with 0.1 mg/L–1 g/L (w/v) CuCl₂ at pH 3, 4, and 5 and 0.01–7.5% (v/v) H₂O₂ at pH 3, 4, and 5 for 3 h (5 mL/cm² dermis). dCELL human dermis was treated with a combination of CuCl₂ and H₂O₂ at a concentration range of 0.1–10 mg/L and 0.01–1%, respectively, between pH 3.1–4.9 for 3 h. All treatments were carried out at 25°C at 200 rpm. After chemical treatment, dCELL human dermis was washed in distilled water (Baxter) thrice for 20 min and frozen at −40°C until further use. Control tissue was nontreated dCELL human dermis.

Histology

After CuCl₂ and H₂O₂ treatments, a sample of dCELL human dermis (∼1 cm²) was cut and fixed in 10% formal saline (VWR). Dermis was embedded in paraffin wax and 5-μm sections were cut using a microtome (Finesse 325; Thermo Scientific) and mounted on slides. Sections were stained with hematoxylin and eosin (H & E) for general morphology. Van Giesons staining was performed for visualization of collagen fibers.

Collagen birefringence

Wax-embedded CuCl₂ and H₂O₂-treated dermis were mounted on glass microscope slides. Sections were dewaxed in xylene (VWR) and dehydrated in ethanol (VWR). Sections were placed in Harris hematoxylin for 8 min followed by picro-sirius red solution (Direct Red 80; Sigma Aldrich) for 1 h. Slides were washed in acidified and distilled water before mounting with DPX (VWR). Sections were viewed under polarizing light microscopy (Leica DMLA). Upon polarization, the stained collagen fibers disperse light displaying colors ranging from green (thinner fibers) to red (thicker, mature fibers). To quantify the proportion of fibers displaying red interference, using ImageJ software, the background was subtracted to allow the total tissue area to be measured (in pixels). The hue was resolved to select the red fibers, which was calculated as a percentage of the total tissue area (in pixels).

Collagen type IV immunohistochemistry

Wax-embedded sections were mounted on silanized coated slides (Dako) and dewaxed by dipping into xylene followed by rehydration in absolute ethanol, 95% ethanol, and distilled water. Sections underwent heat-induced epitope retrieval in an antibody retrieval solution (Dako) at 95–99°C for 5 min. The endogenous peroxidase activity was quenched by incubation with peroxidase block solution (Dako). Sections were incubated with monoclonal mouse anti-human collagen IV antibody (Dako) for 60 min. Sections were then incubated with peroxidase-labeled polymer for 30 min followed by incubation with diaminobenzidine (DAB⁺) substrate chromagen solution (Dako). To ensure there was no nonspecific binding of the primary antibody, negative control tissue was treated with IgG (Dako). All sections were viewed with a Leica DMLA light microscope.

Biomechanical analysis–ultimate tensile strength

Lloyds Instruments Universal Testing Machine (LRX Plus) was used to determine the structural strength of cellular and dCELL dermis treated with CuCl₂ and H₂O₂. Tissue was cut into universal dumbbell shapes (35-mm length ×2-mm minimal width) using a Ray Ran die cutter (DIN-5304 Type S3). The thickness of the dermis was measured using Digimatic Vernier calipers (Mitutoyo). Dumbbells were pulled to breaking point with a 100N load cell at a speed of 100 mm/min. Ultimate tensile stress (N/m²) was determined by dividing load (Newtons) at break by the cross-sectional area, using Nexygen software.

Biochemical analysis–collagen denaturation

Five millimeter biopsies from CuCl₂- and H₂O₂-treated dermis were treated with 10 mg/mL α-chymotrypsin (Sigma Aldrich) for 24 h (37°C) to digest denatured collagen, releasing hydroxyproline. Positive and negative controls were achieved by treating dermis with 2.5 M sodium hydroxide and 50 mM glutaraldehyde, respectively, for 16 h (25°C). The supernatant was acid hydrolyzed (12 M HCl; 121°C and 18 psi for 1 h) followed by treatment with chloramine T (Sigma Aldrich) and 4-(Dimethylamnio)-benzaldehyde (Sigma Aldrich). The level of hydroxyproline in the supernatants was obtained by measuring the absorbance at 595 nm on an Ultra Microplate reader (BioTek).

In vitro cytotoxicity

Five millimeter biopsies of CuCl₂- and H₂O₂- treated dermis were attached to the base of wells of a 24-well tissue culture plate using steristrips (3M Healthcare). Human skin fibroblasts (HSF) and MG63 cells (an osteosarcoma cell line; ECACC) were grown to 70% confluency, harvested, counted, and seeded into the wells containing dermis and incubated for 24 h in a 5% CO₂ and 37°C incubator. Cells were fixed with 50%, 70%, and 100% ethanol and stained with 20% Giemsa solution (VWR). Growth of cells up to and around the skin biopsies was visualized using an inverted microscope (Leica, 090-135-002).

Statistical analysis

All numerical data were analyzed using GraphPad Prism 5 software. All numerical data are shown as mean values ±95% confidence limits. Statistical significance was assessed using one-way analysis of variance (ANOVA). Values of p < 0.05 were considered to be statistically significant.

Results

Stability of CuCl₂ and H₂O₂ solutions

Solutions of 0.1 mg/L CuCl₂ were below the lower sensitivity threshold of the assay; so the stability of this solution was not tested. In solutions containing either only CuCl₂ (1 mg/L–1 g/L) or only H₂O₂ (0.01–7.5%), there was no change from the initial concentrations of either reagent over a 44-day period, irrespective of the pH (data not shown).

When combinations CuCl₂ and H₂O₂ were mixed (Day 0), there was a significant reduction in the detectable concentration of copper, within minutes, in 1, 10, and 100 mg/L CuCl₂ solutions with 7.5% H₂O₂ (Fig. 1 and Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/tec). Similarly, a three-fold reduction in copper concentration was measured when 1 mg/L CuCl₂ was mixed with 3% H₂O₂ (Fig. 1A and Supplementary Table S1). After 14 days, a decline in the concentration of 10 mg/L CuCl₂ was evident when mixed with 3% H₂O₂ (Fig. 1B and Supplementary Table S1). When 100 mg/L CuCl₂ was combined with 3% H₂O₂, the level of detectable copper remained at a relatively constant level, however, significant changes in concentration were detected on individual days (Fig. 1C and Supplementary Table S1).

FIG. 1.

Concentration of Cu²⁺ ions in solutions of 1 mg/L (A), 10 mg/L (B), and 100 mg/L CuCl₂ (C) when combined with H₂O₂ in the concentration range of 0.01–7.5% over 44 days. n = 5. *p < 0.05, **p < 0.01, ***p < 0.001 Cu²⁺ ion concentration in solutions containing H₂O₂ versus Cu²⁺ ion concentration in solutions without H₂O₂ at specified time points. Color images available online at www.liebertpub.com/tec

Over 21 days, all concentrations of H₂O₂ (0.01–3%) were maintained in the presence of 0.1, 1, and 10 mg/L CuCl₂ (Fig. 2). However, an increase in CuCl₂ to 100 mg/L demonstrated a significant reduction in the measured concentrations of 0.5%, 1%, and 3% H₂O₂ solutions from 14 days, which continued to decline over a further 30 days (Fig. 2C–E and Supplementary Table S2). The pH of solutions containing a mixture of CuCl₂ and H₂O₂ was maintained between pH 3–5 over a 44-day period and therefore there was no need to adjust the pH (Table 1).

FIG. 2.

Concentration of H₂O₂ in solutions of 0.01% (A), 0.1% (B), 0.5% (C), 1% (D), and 3% H₂O₂ (E) when combined with CuCl₂ in the concentration range of 0.1–100 mg/L over 44 days. n = 5. *p < 0.05, **p < 0.01 H₂O₂ concentration in solutions containing CuCl₂ versus H₂O₂ concentration in solutions without CuCl₂ at specified time points. Color images available online at www.liebertpub.com/tec

Based on these findings, the data presented in this article is of dCELL dermis treated with solutions containing 0.1 mg/L–1 g/L (w/v) CuCl₂ or 0.01–7.5% (v/v) H₂O₂ at pH 3, 4, and 5. dCELL human dermis was also treated with combinations of CuCl₂ in the concentration range of 0.1, 1, and 10 mg/L together with H₂O₂ at concentrations of 0.01%, 0.1%, 0.5%, and 1%.

Histological analysis

H&E staining of decellularized human dermis treated with 0.1 mg/L–1 g/L CuCl₂ between pH 3–5 and 0.01–7.5% H₂O₂ between pH 3–5 showed no differences in gross histology compared to nontreated dCELL human dermis (data not shown). Similarly, there were no differences in the extracellular matrix structure when dCELL human dermis was treated with a mixture of CuCl₂ and H₂O₂ up to the maximum concentrations of 10 mg/L and 1.0%, respectively, (Fig. 3).

FIG. 3.

H & E staining of dCELL human dermis (A) and dCELL human dermis treated with the maximum combined concentration of CuCl₂ (10 mg/L) with H₂O₂ (1%) (B). Original magnification ×20. Color images available online at www.liebertpub.com/tec

Ultimate tensile strength

There was no significant difference in the ultimate tensile strength of dCELL human dermis treated with 0.1 mg/L–1 g/L CuCl₂ or 0.01–7.5% H₂O₂ compared to nontreated dCELL human dermis at pH 3, 4, and 5 (Fig. 4A, B). Similarly, 0.1 mg/L CuCl₂ combined with all concentrations of H₂O₂ and 1 mg/L CuCl₂ combined with 0.01%, 0.1%, and 0.5% H₂O₂ did not affect the ultimate tensile strength of dCELL human dermis (Fig. 4C). However, addition of 1.0% H₂O₂ to solutions of 1 mg/L CuCl₂ resulted in a significant increase in the ultimate tensile strength of dCELL human dermis (Fig. 4C). A 10-fold increase in CuCl₂ to 10 mg/L combined with H₂O₂ (0.1–1%), however, did not result in any significant change in dCELL dermis ultimate tensile strength (Fig. 4C).

FIG. 4.

Ultimate tensile strength of dCELL human dermis treated with 0.1 mg/L–1 g/L CuCl₂ (A) or 0.01–7.5% H₂O₂ (B) at pH 3, 4, and 5 and 0.1–10 mg/L CuCl₂ combined with 0.01–1% H₂O₂ (C). Ultimate tensile strength of nontreated dermis was 18.52 N/mm² ± 2.44. *p < 0.05 1 mg/L CuCl₂ + 1% H₂O₂-treated dermis versus nontreated dCELL human dermis. Error bars are 95% CI of the means.

Collagen analysis

To assess if treatment of dCELL human dermis with CuCl₂ and H₂O₂ caused any damage to the collagen fibers, Van Giesons stain, collagen birefringence, and collagen type IV immunohistochemistry was performed. In addition, any denaturation of collagen fibers was evaluated by measuring hydroxyproline levels in solubilized dCELL human dermis.

Collagen appeared tightly packed and uniformly organized; Van Giesons staining showed that CuCl₂ in the concentration range of 0.01–10 mg/L together with H₂O₂ in the concentration range of 0.01–1.0% caused no visual disruption to the collagen network (Fig. 5). In addition, to identify if there was any loss of mature collagen fibers, dermis was stained with picro-sirius red and viewed under polarized light resulting in birefringence of the collagen fibers in which mature collagen fibers were viewed as red in color, which were quantified as a percentage of the total tissue area. There were no significant differences in the quantity of stained mature collagen fibers of dCELL dermis treated with single and combined CuCl₂ and H₂O₂ solutions (Tables 2 and 3).

FIG. 5.

Van Giesons staining of dCELL human dermis (A) and dCELL human dermis treated with the maximum combined concentration of CuCl₂ (10 mg/L) with H₂O₂ (1%) (B). Original magnification ×20. Color images available online at www.liebertpub.com/tec

Table 2.

The Percentage of Red Interference of CuCl₂- (0.1 mg/L, 10 mg/L, and 1 g/L) and H₂O₂- (0.01%, 0.1%, and 7.5%) Treated dCELL Human Dermis Stained with Picro-Sirius Red, Viewed Under Polarized Light Microscopy

	% red interference
	55.18 ± 0.82
dCELL dermis	pH 3	pH 4	pH 5
0.1 mg/L CuCl₂	60.85 ± 6.32	55.65 ± 0.46	47.41 ± 2.12
10 mg/L CuCl₂	57.00 ± 0.96	50.14 ± 8.27	47.51 ± 10.76
1 g/L CuCl₂	63.89 ± 5.08	61.37 ± 3.06	57.87 ± 6.10
0.01% H₂O₂	51.26 ± 5.08	53.69 ± 1.00	40.58 ± 3.58
1% H₂O₂	63.48 ± 3.77	49.85 ± 3.60	47.85 ± 12.12
7.5% H₂O₂	55.18 ± 1.21	56.49 ± 0.16	56.70 ± 8.05

Red interference of nontreated dCELL dermis was 55.18% ± 0.82. There was no significant difference in the percentage of red interference of treated dermis compared to nontreated dCELL dermis. Values are means ±95% CI.

Table 3.

The Percentage of Red Interference of dCELL Human Dermis Treated with Combined Mixtures of CuCl₂ (0.1, 1, and 10 mg/L) and H₂O₂ (0.1%, 0.1%, 0.5%, 1%) that Have Been Stained with Picro-Sirius Red and Viewed Under Polarized Light Microscopy

	% red interference
0.1 mg/L CuCl₂
0.01% H₂O₂	51.84 ± 1.80
0.1% H₂O₂	53.44 ± 2.20
0.5% H₂O₂	56.47 ± 0.40
1% H₂O₂	59.60 ± 8.80
1 mg/L CuCl₂
0.01% H₂O₂	56.62 ± 6.39
0.1% H₂O₂	59.31 ± 1.59
0.5% H₂O₂	48.80 ± 10.38
1% H₂O₂	59.45 ± 19.37
10 mg/L CuCl₂
0.01% H₂O₂	60.67 ± 1.63
0.1% H₂O₂	55.08 ± 1.53
0.5% H₂O₂	61.03 ± 0.91
1% H₂O₂	62.05 ± 1.09

There was no significant difference in the percentage of red interference of treated dermis compared to nontreated dCELL dermis (55.18% ± 0.82). Values are means ±95% CI.

Collagen type IV immunostaining was primarily located in the basement membrane of the dermal–epidermal junction and blood vessels (Fig. 6). Immunohistochemical staining showed that collagen type IV remained intact after treatment of dCELL human dermis with 0.1 mg/L–1 g/L CuCl₂ (pH 3, 4, and 5), 0.01–7.5% H₂O₂ (pH 3, 4, and 5), or combinations of 0.1 mg/L CuCl₂ with 0.01–1% H₂O₂ and 1 mg/L CuCl₂ with 0.01–1% H₂O₂ (Fig. 6). An increase in CuCl₂ concentration to 10 mg/L together with 1% H₂O₂ caused staining for collagen type IV to be lost at the epidermal–dermal junction; however, positive staining was still seen at the basement membrane of blood vessels of these samples (Fig. 6).

FIG. 6.

Collagen IV immunohistochemical staining of dCELL human dermis treated with CuCl₂ in combination with H₂O₂ at the concentrations stated. Loss of collagen IV staining was evident at the dermal–epidermal junction of dCELL dermis treated with 10 mg/L CuCl₂ + 1% H₂O₂ (black arrow). Negative control tissue was treated with IgG. Original magnification ×20. Color images available online at www.liebertpub.com/tec

Treating dCELL dermis with CuCl₂ (1 mg/L–1 g/L), H₂O₂ (0.01–7.5%) at pH 3, 4, and 5 and 0.1 mg/L, 1 mg/L, and 10 mg/L CuCl₂ in combination with 0.01–1% H₂O₂ did not induce a significant change in hydroxyproline release compared to nontreated dCELL dermis (Fig. 7).

FIG. 7.

Hydroxyproline release from dCELL human dermis treated with 0.1 mg/L–1 g/L CuCl₂ (A), 0.01–7.5% H₂O₂ (B), and combined solutions of 0.1–10 mg/L CuCl₂ with 0.01–1% H₂O₂ (C). Hydroxyproline from nontreated dCELL human dermis was 1.68 μg/mg ±0.71. Error bars are 95% CI of means.

Biocompatibility

The combination of 10 mg/L CuCl₂ with 1% H₂O₂ was observed to induce some cytotoxicity in HSF cells with sparse growth around the dCELL dermis (Fig. 8). All other combinations were not cytotoxic to either HSF or MG63 cells (not shown).

FIG. 8.

In vitro contact cytotoxicity of combined CuCl₂- and H₂O₂ -treated dCELL human dermis with HSF, original magnification ×10. HSF grew around and in contact with dCELL human dermis treated with all concentrations and combinations of CuCl₂ and H₂O₂ except for 10 mg/L CuCl₂ with 1% H₂O₂ where HSF made sparse contact with the dCELL dermis. HSF grew when cultured with steristrips (negative control). There was no evidence of growth around the cyanoacrylate glue (positive control). Color images available online at www.liebertpub.com/tec

Discussion

The purpose of this study was to determine the concentrations of CuCl₂ and H₂O₂, either singly or in combination, which did and did not elicit changes in morphology, functionality, and biocompatibility of dCELL human dermis. Concentrations of CuCl₂ were chosen, which are below the toxic levels for humans–mean plasma copper concentration, in a healthy adult range between 80–100 mg/dL (800–1000 mg/L).³⁵ Unbound copper ions exhibit greater toxicity compared to complexed copper.³⁶ H₂O₂ lacks toxicity following its rapid decomposition to H₂O and O₂, making it a suitable decontamination reagent where human application is concerned. For example, 3–6% (v/v) H₂O₂ is commonly used as an antiseptic and a contact lens cleaning solution, dental disinfectants often contain up to 1% H₂O₂, and 3% H₂O₂ is used routinely during processing of human bone.^28,29,32,37 In this study, concentrations up to 7.5% (v/v) H₂O₂ were found to have no detrimental effect on the structural or functional properties of dCELL human dermis. pH, temperature, and metal ions all affect the stability of H₂O₂. H₂O₂ is stable at low pH; so in this study, all solutions containing H₂O₂ were made between pH 3–5. Increases in temperature accelerate the decomposition of H₂O₂. While a rise in ambient temperature to 30°C has little effect on H₂O₂ stability, every further 10°C increase in temperature causes approximately a 2.2-fold increase in the rate of decomposition.³⁸ In this study, all treatments were performed at 25°C. Metal ions, particularly manganese, iron, and copper also have a negative effect on the stability of H₂O₂. The stock H₂O₂ used in this study contained stabilizers in the form of metal chelators. At 3% and 7.5% H₂O₂, these chelators may have been in sufficient quantity to chelate the copper ions (Fig. 1). In addition, any free copper ion at the higher CuCl₂ concentrations would have induced breakdown of H₂O₂, explaining the reduction in the amount of measurable H₂O₂ (Fig. 2).

Histological visualization revealed that single and combined CuCl₂ and H₂O₂ treatment did not cause any alteration to the morphological architecture of dCELL dermis. In this study, dermis was obtained from donors over the age of 50 years. The collagen fibers appeared tightly packed, uniformly aligned, and thicker, presumably mature fibers were evident in the reticular layer. Neither the single or combined treatments of CuCl₂ and H₂O₂ had any effect on collagen structure or orientation. Hydroxyproline is a major component of collagen, having a key role in stabilizing the triple-helical collagen structure.³⁹ Measuring free hydroxyproline levels indicates the proportion of denatured collagen. In this study, CuCl₂ and H₂O₂ treatments were not associated with any alteration in hydroxyproline content. Collagen type IV is a major component of the basement membrane at the dermal–epidermal junction where it maintains structural integrity and functionality of the membrane as well as having an important role in wound healing.^40–42 NHSBT produces dCELL dermis for topical applications such as treating burn injuries and nonhealing ulcers and for internal injuries such as rotor cuff injuries. Reepithelialization of the decellularized graft is an essential part of the wound healing process when used topically.¹⁷ Collagen type IV has a key role in inducing keratinocyte migration to the wound site, an early and crucial part of the reepithelialization process.⁴³ A loss in collagen type IV immunoreactivity at the dermal–epidermal junction of dermis was observed with the maximum combined concentration of CuCl₂ (10 mg/L) and H₂O₂ (1%). Oxidative damage to collagen is well documented. Metal ions, notably cuprous [copper (I)] and cupric ions when mixed with H₂O₂ mediate site-specific damage through binding to collagen, resulting in cross-linking, decarboxylation, and fragmentation of collagen.⁴⁴ These mechanisms may have accounted for this loss of collagen type IV staining.

Biomechanical properties of skin depend on the body location. Collagen and elastin are responsible for the tensile strength and viscoelasticity of skin, respectively. Changes in the orientation of dermal collagen fibers during deformation minimize the strain of the skin.⁴⁵ In this study, tensile strength of dCELL dermis was measured by application of a constant slow increase in load (100N at 100 mm/min). Treating dCELL human dermis with CuCl₂ or H₂O₂ individually did not compromise the ultimate tensile strength. However, 1 mg/L CuCl₂ with 1% H₂O₂ induced a significant increase in ultimate tensile strength. Interestingly, a further increase in CuCl₂ to 10 mg/L with H₂O₂ did not have a significant effect on the dCELL dermis ultimate tensile strength. No denaturation of collagen occurred with CuCl₂ and H₂O₂ individual treatments, which may account for the maintenance of the ultimate tensile strength

After CuCl₂ and H₂O₂ treatments, dCELL human dermis was washed with distilled water to remove any residual reagents. In vitro contact cytotoxicity identified that the maximum combined concentration of CuCl₂ (10 mg/L) and H₂O₂ (1%) had slight cytotoxic effects on HSF. The cytotoxic effect of copper ions on mouse skin fibroblasts has previously been reported by Cao et al. (2012).⁴⁶

This study aimed to identify the maximum concentration range of CuCl₂ and H₂O₂ that, when applied to dCELL human dermis would not have an adverse effect on the tissue. The data indicate that single treatment of CuCl₂ in the concentration range of 0.1 mg/L–1 g/L or a single treatment of H₂O₂ in the concentration range of 0.01–7.5% had no effect on the morphology of the acellular scaffold. The collagen network remained intact, consistent with the hydroxyproline analysis that suggested there was no collagen denaturation associated with these treatments and the biomechanical data that showed there was no change in the tensile strength. The treated dermis was biocompatible in vitro with both primary cells and a cell line, having no effect on their morphology or proliferation. Combining both 10 mg/L CuCl₂ with 1% H₂O₂ did cause damage to the decellularized dermal scaffold; notably, there was a loss of collagen type IV at the dermal–epidermal junction and induced cytotoxicity in primary HSF. Based on the findings presented here, application of CuCl₂ up to a concentration of 1 g/L or H₂O₂ up to 7.5% individually or combining 10 mg/L CuCl₂ with 0.5% H₂O₂ are the maximum doses of these chemicals that do not have an adverse effect in terms of morphology, functionality, and biocompatibility. Further work should investigate the effects of these treatments on other tissue allografts and the bactericidal effects of the combined reagents.

Footnotes

Acknowledgment

We acknowledge the support of NHSBT for the funding of this research.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Evaluation of Copper and Hydrogen Peroxide Treatments on the Biology,Biomechanics,and Cytotoxicity of Decellularized Dermal Allografts

Abstract

Introduction

Methods

Tissue

Dermis decellularization

Stability of copper chloride and hydrogen peroxide solutions

Copper chloride and hydrogen peroxide treatment

Histology

Collagen birefringence

Collagen type IV immunohistochemistry

Biomechanical analysis–ultimate tensile strength

Biochemical analysis–collagen denaturation

In vitro cytotoxicity

Statistical analysis

Results

Stability of CuCl2 and H2O2 solutions

Histological analysis

Ultimate tensile strength

Collagen analysis

Biocompatibility

Discussion

Footnotes

Acknowledgment

Disclosure Statement

References

Supplementary Material

Stability of CuCl₂ and H₂O₂ solutions