A Histological and Biomechanical Analysis of Human Acellular Dermis (HAD) Created Using a Novel Processing and Preservation Technique

Abstract

Background

Large and complex defects requiring reconstruction are challenging for orthopaedic surgeons. The use of human acellular dermal (HAD) matrices to augment large soft tissue defects such as those seen in massive rotator cuff tears, knee extensor mechanism failures and neglected Tendo-Achilles tears has proven to be a valuable tool in surgeons reconstructive armamentarium. Different methods for allograft decellularization and preservation alter the native properties of the scaffold. Traditional processing and preservation methods have shown to have drawbacks that preclude its widespread use. Some of the common issues include inferior biomechanical properties, the risk of rejection, limited customization, difficulty in storing and transporting, the requirement of pre-operative preparation, and last but not the least increased cost.

Methods

We describe a novel processing and preservation method utilizing a two-step non-denaturing decellularization method coupled with preservation using a water-sequestering agent (glycerol) to remove immunogenic components while retaining biomechanical properties. The efficiency of this novel process was compared with the traditional freeze-drying method and verified by histological evaluation and biomechanical strength analysis.

Results

The absence of cellular components and matrix integrity in hematoxylin and eosin-stained glycerol-preserved HAD (gly-HAD) samples compared to freeze-dried HAD (FD-HAD) demonstrated effective yet gentle decellularization. Biomechanical strength analysis revealed that gly-HADs are stronger with an ultimate tensile load to the failure strength of 210 N compared to FD-HAD (124N). The gly-HADs were found to have an optimal suture–retention strength of 126 N. Finally, sterility testing of the resultant grafts was checked to ensure a sterility assurance level of 10⁻⁶ to establish implantability.

Conclusion

The novel processing and preservation technique is described in this paper to create a Human Acellular Dermis with higher biomechanical strength and superior histological characteristics. The processing and preservation technique ensured high sterility assurance levels to establish implantability.

Introduction

Dermal allografts are derived from cadaveric human skin and are decellularized pieces of the dermis that are processed and sterilized using various techniques. These allografts offer the versatility to repair numerous reconstructive defects. The ever-increasing and aging yet active population represents increased incidences of rotator cuff tears, large tendon ruptures, and burn injuries. The need for enhanced outcomes of biological augmentations in reconstructive surgeries necessitates the development of safe, effective, and affordable allograft choices. Human skin is the largest organ of the body with an average surface area of 30 m² in adults [1]. 70–80% of the skin is majorly composed of collagen typically type I, III, and a minor component of elastin [2]. Native human dermis harbours blood vessels, nerve endings, hair follicles, glands, and dermal cells.

An ideal extracellular matrix (ECM) scaffold should provide the native biological and mechanical cues for tissue regeneration allowing the host cells to migrate into it and incorporate themselves into the graft. Eliminating native immunogenic components from the human dermis results in an ECM composed of collagen, elastin, growth factors, and proteoglycans. Human acellular dermal graft is often used to capitalize on the native ECM properties that serve as a scaffold for host cell infiltration followed by gradual remodeling of the tissue matrix leading to revascularization, lymphangiogenesis, and finally integration with the host tissue [3]. The allograft is intended for use as an “onlay” on top of a repair or as a bridge between tissues for various reconstructive surgeries including but not limited to burn injuries, orthopedic surgeries, plastic surgery, chronic wounds, oral surgery, abdominal surgery, otolaryngology [4]. The use of these grafts eliminates or decreases the need for an autograft whose use is often criticized for additional surgical morbidity and time.

An acellular dermal graft is typically devoid of cellular components that cause an exuberant inflammatory response. Unlike porcine/bovine intestinal submucosa and urinary bladder matrix; considering the thickness, density, and complexity of the human dermis, a gentler decellularization process is to be employed to maintain the integrity of the dermal ultrastructure while retaining biomechanical strength.

The aim of the present study is to describe a novel technique for creating an acellular human dermal allograft. The study also assesses the suitability of this graft for transplantation into a human by ensuring optimal strength, low risk of disease transmission, and elimination of cellular immunogenic components.

Materials and Methods

Overview of Experimental Design

Acellular dermal grafts were derived from human full-thickness skin graft (FTSG) through a regulated process derived in compliance with the country laws of The Transplantation of Human Organs and Tissues Act (THOTA) 1994 [5] and state Guidelines for Tissue Banking by Regional cum State Organ and Tissue Transplant Organization (ROTTO–SOTTO) Mumbai, 2021 [6]. More than 10 acellular dermal graft patches were produced from skin donations by 03 deceased donors in the period between January 2023 and April 2023.

Key processes involved in HAD samples, as highlighted in Fig. 1.

Fig. 1.

Human acellular dermal graft processed using a novel technique (gly-HAD) from retrieved human full-thickness skin. Parts of the figure were drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0)

Tissue Acquisition

Human full-thickness skin grafts (FTSGs) were retrieved from brain-dead and/or circulatory dead donors with prior written informed consent from the next-of-kin. The deceased donors were evaluated based on the eligibility criteria (Table 1) for contraindications before proceeding with the retrieval. Adherence to stringent screening criteria is a cardinal step to ensure recipient safety against infection and disease transmission. Blood samples for deceased donor screening were taken at the time of procurement and tested for Human Immunodeficiency Virus Antibodies (HIV-1/2-Ab), Hepatitis B Virus Surface Antigen (HBsAg), Hepatitis C Virus Antibodies (HCV-Ab) [6].

Table 1.

Donor eligibility criteria set forth for skin retrieval in compliance with the guidelines of state-appropriate authority [6]

Donor inclusion criteria

Donor exclusion criteria

Donor is brain stem dead and certification of brain stem death and death certificate is available Donor is circulatory dead and their death certificate is available The donor’s treating registered medical practitioner has evaluated and approved the donation process The donor’s next-of-kin is informed about skin donation and they have provided written consent for skin donation Donor age is between 18 to 75 years, any gender Donor’s skin retrieval is scheduled within 6 h of asystole

History, or presence of, or suspicion of, or risk factors for the following Hepatitis B or C, HIV 1 or 2, Septicemia, systemic viral disease or mycosis, or active tuberculosis Central degenerative neurological diseases of possible infectious origin Autoimmune or connective tissue disease Use of all native human pituitary-derived hormones History of dura-mater allograft, including unspecified intracranial surgery Malignancy Any immunosuppressive treatment Exposure to a toxic substance that may be transferred in toxic doses or damage the tissue Infection or prior irradiation at the site of donation COVID in the last 1 month Unknown cause of death

The eligible donors were prepped for skin retrieval in the hospital operating room following aseptic techniques. Skin grafts are preferably retrieved from the back of the thighs, buttocks, back region, and lateral calf region. The retrieval technique and the retrieval site have a considerable effect on the final quality of the graft. The current study involved the retrieval of skin from the calf region by the retrieval team’s registered medical practitioner/trained technician. The donor site was cleaned and shaved followed by betadine scrub and chlorhexidine painting. Clean incisions were made through the donor skin using surgical blades and full-thickness skin patches were separated from the subcutaneous tissue. The donor site was wrapped in appropriate dressing with gamjee pads, bandage rolls, and absorbent sheets so as to not hamper the funeral rituals.

Transportation

The retrieved skin was stored in sterile polyethylene terephthalate containers (PET) with a sterile physiological solution and shipped to the tissue bank under 0–10 °C.

Preprocessing

Upon arrival at the tissue bank, the donor skin was removed from the transportation container in the biosafety cabinet and placed with its reticular side down on a clean sterile stainless-steel tray and cut into rectangular patches with sizes ranging from 3 × 5 cm to 10 × 10 cm. A small cut was made on the bottom right corner of the graft to distinguish the reticular and papillary surfaces after processing. The grafts were then stored at 0–10 °C in a physiological solution with antibiotics prior to processing. Antibiotics are an essential step in the storage due to the presence of collagen-degrading proteases from existing bioburden [7]. Tissues were kept at these low temperatures for a maximum of 7 days until processing.

Processing

A novel process of a two-step mild chemical decellularization technique in combination with a hygroscopic solvent preservation method was employed. The novel preservation step was further compared with the traditional freeze-drying technique to assess its efficiency.

Decellularization

Tissues from each donor were processed separately avoiding any cross-contamination due to inherent bioburden and/or human error. The FTSGs were cleaned of any subcutaneous fat by gentle mechanical scraping followed by treatment with decellularizing reagents. The grafts were denuded of the cellular components using a two-step process of hypertonic solution to remove the epidermis (Fig. 2) followed by mild anionic detergent treatment [8]. Decellularization was followed by thorough rinsing in physiologic solution accompanied by agitation and /or ultrasonication to remove residual decellularizing reagents.

Fig. 2.

Processing of human full-thickness skin graft: a human full-thickness skin graft after cutting, b dermal graft after processing, c gly-HAD ready to be packaged

Preservation and packaging

There are various preservation techniques available to maintain the grafts’ shelf life for as long as 5 years [9]. In this study, two preservation methods and their effects on the final biomechanical properties of the grafts were evaluated.

Freeze drying

Preservation by freeze-drying involves the removal of water from the specimen under a vacuum leading to the final specimen with reduced water activity facilitating increased shelf life at room temperature. The decellularized grafts were disinfected with 70% isopropyl alcohol and stored at − 80 °C for 18–24 h followed by the drying cycle in Martin Christ Alpha 1–4 LSC Plus. The program for the lyophilizer was set, such that the final residual moisture content of the grafts after freeze-drying was 2–6%. The freeze-dried human acellular dermis (FD-HAD) grafts were then heat sealed in impervious pouches and stored at room temperature not exceeding 35 °C followed by terminal sterilization.

Glycerol preservation

The decellularized grafts were disinfected with 70% isopropyl alcohol and exposed to a preservation solution for 1 h at 37 °C. Glycerol was chosen as the preserving solution in the present study attributing to its unique hygroscopicity in addition to non-toxicity, compatibility with tissues, low molecular weight, and is generally recognized as safe by the FDA [10, 11]. Excess reagents were removed by dry spinning and/or blotting followed by packaging of the glycerol-preserved HAD (gly-HAD) under aseptic conditions in inert packages and stored at room temperature not exceeding 35 °C followed by terminal sterilization.

Prior to packaging, representative samples from each donor's dermal grafts were taken for quality assurance and quality control evaluation.

Quality Assurance

Histological evaluation: In the present study, it was hypothesized that the dermal graft processing steps should render the tissue acellular along with intact collagen network; to verify the same, conventional histological techniques were used. Hematoxylin and eosin staining (H & E) is a common method used to provide a clear microanatomy of the tissues [12,13]. Dermal tissue samples were fixated with 10% formalin solution. After processing and sectioning, histological cross sections were stained with the H & E method and visualized under 10× & 40× magnification. Tissue histology of control (unprocessed FTSG), gly-HAD, and FD-HAD were compared for remnants of cellular components and collagen banding.

Bioburden Testing

The Bioburden of the specimen is the number of viable microorganisms present in the specimen after the completion of processing steps and prior to sterilization [13]. It is also known as the ‘contamination level’ of the specimen [14]. Bioburden testing is a crucial step to assess the efficiency of the processing method. It is also important for validation and substantiation of sterilization dose to achieve implantable tissue graft. Vacuum filtration technique is an accepted method of bioburden evaluation by ISO 11737-1 standards [15], to ensure validated sterilization doses are derived to render the dermal graft sterile. In the present study, microbiological analysis of processed glycerol preserved non-radiated dermal sample (gly-HAD) was performed using a vacuum filtration technique followed by incubation of the membrane in Tryptic soy agar for 14 days at 30°C to 35°C.

Sterilisation

The packaged processed dermal grafts were maintained at − 78.5 °C using dry ice and subjected to a 25kGy dose of gamma radiation from a ⁶⁰cobalt source. Low-temperature treatment was employed to prevent the collagen fibril damage associated with gamma radiation [16]. The sterilization dose was derived by the VD²⁵max approach after analysis of the bioburden results for levels of average bioburden ≥1000 CFU and verification sterility assurance level (SAL) = 10^-6.

Quality Control

Sterility Testing

Processed glycerol preserved dermal samples gamma radiated with 25kGy dose were tested for the presence of viable microorganisms using recommendations in ISO 11737-2 [17]. The radiated samples were incubated in sterile Tryptic soy broth at 30–35 °C for 14–21 days to validate the sterilization dose as per ISO 11137-2 [18].

Biomechanical Testing

The ideal acellular dermal grafts should be capable of bearing anisotropic load as required in their clinical applications [4]. The load-bearing capacity of an allograft depends upon the integrity and flexibility of the collagen matrix [16]. To assess the biomechanical properties, the ultimate tensile load was determined using the UTM—Universal Testing Machine (Tinius Olsen Universal Testing Machine with a calibrated load cell of 5KN) [19]. The biomechanical testing was focused on the strength of the dermal samples and no control was used. Each dermal sample length, width, and thickness were individually recorded using digital calipers. The measured specimens were pressure clamped onto the UTM grippers and were pulled at a rate of 100% strain per second until failure. The load-to-failure portion of the test graph was used to determine the ultimate tensile force.

An irradiated gly-HAD sample with dimensions (length × width × thickness) 100 × 17 × 1.2 (mm) was clamped onto the UTM for testing (Fig. 3a). Irradiated FD-HAD sample with dimensions 100 × 17 × 1.2 (mm) was removed from the sterile packaging and rehydrated with 0.9% saline at room temperature for 15 minutes before testing followed by clamping onto the UTM grippers.

Fig. 3.

Biomechanical evaluation of processed glycerol preserved dermal samples on Universal testing machine. a Irradiated gly-HAD. b Single-ended sutured gly-HAD. c Double-ended sutured gly-HAD

To estimate the suture retention strength, the materials were removed from their sterile packages at the time of testing and cut into strips of 60 mm length and 15mm wide. A single vertical stitch of No. 2 FiberWire was passed through the dermal sample at a distance of 10 mm from one end (Single end sutured gly-HAD) and clamped onto the UTM for suture pull-out strength (Fig. 3b). To stimulate clinically relevant configuration, another sample was tested where a single vertical stitch of No. 2 FiberWire was passed through the dermal sample at a distance of 10 mm from both the ends (double end sutured gly-HAD) as shown in Fig. 3c.

Results

Quality Assurance

Histological Evaluation

Light microscope analysis of H&E-stained dermal samples mapped out the clear histological differences in processing methods, as shown in Fig. 4. Scanning across the control unprocessed FTSG sample (Fig. 4a, b) delineated the epidermis with numerous purple-stained nuclear materials embedded in pink collagen network. The epidermis was followed by a thick horizontal collagen network of papillary dermis progressing into a thicker network of reticular dermis. On the contrary, the gly-HAD sample (Fig. 4c, d) showed the absence of an epidermis with a pink-stained collagen network devoid of any nuclear remains presenting the acellular nature. The collagen fibrils were observed as intact intricate patterns arranged in a horizontal fashion similar to that of the control indicating possible retained flexibility and strength. FD-HAD sample (Fig. 4e) showed a pink-stained collagen fiber network reoriented from its normal horizontal pattern with the absence of cellular components. Though acellular in nature, freeze drying yielded dermal grafts which were more brittle and less flexible than native dermal grafts.

Fig. 4.

Histological evaluation of hematoxylin and eosin-stained dermal samples showing preserved acellular structure post-processing in c, d gly-HAD sample, while e FD-HAD sample demonstrates acellular matrix with asymmetrical network re-oriented from the a, b control: native structure

Bioburden Testing

Dermal graft from a mammalian origin is inherently characterized by numerous bacteria, spores, and fungi [1]. Variations in the microbiological quantity of the processed graft can substantially affect the radiation sterilization dose required for the tissue to be suitable for implantation [20]. The average batch bioburden as evaluated was found to be 480 CFU/ graft. Since the average is less than 1000 CFU/ graft and the sterility assurance level is -6, the reference table by ANSI/AAMI/ISO 11137 standards [18] was followed to determine the appropriate applied dose as 25 kGy.

Quality Control

Sterility Testing

The chemical decellularization coupled with radiation is effective in devitalizing a variety of bacteria, spores, and fungi [7]. Implantable medical biomaterials are terminally sterilized to achieve a sterility assurance level (SAL) of 10⁻⁶, which implies a probability of finding not more than one viable microorganism in one million sterilized items of the final graft. Sterility testing results showed 0 CFU from the processed glycerol preserved dermal samples radiated with 25 kGy gamma radiation.

Biomechanical Testing

The irradiated gly-HAD sample exhibited an ultimate tensile force of 210 N before failing whereas the irradiated FD-HAD sample failed at 124N (Fig. 5). The results confirmed the hypothesis that FD-HAD was brittle and had less structural integrity owing to the rearranged collagen network. The suture retention test showed the single-ended sutured gly-HAD failed when the suture pulled through the graft material at 126 N, while the double-ended sutured gly-HAD failed from one end at 102 N (Fig. 5). No suture breakage was observed among the test samples. These results imply that gly-HAD offers sufficient mechanical integrity and effectiveness, which is essential for its successful use in various clinical scenarios. In addition, many other factors influence the biomechanical properties of the dermal graft including but not limited to donor age, gender, and retrieval site; these factors were beyond our scope due to donor information confidentiality.

Fig. 5.

a Ultimate load-to-failure comparison between FD-HAD and gly-HAD, demonstrating FD-HAD is less strong than gly-HAD which has higher mechanical integrity. b Suture retention strength evaluation for single-end and double-end pull-out using FiberWire No. 2

Discussion

The creation of an implantable graft from a human source presents unique complexities from screening, procurement, transportation to tissue bank, processing, preservation, and sterilization all the while considering biologic variability among donors and adhering to ethical practices for tissue donation.

Normal tissue repair involves “dynamic reciprocity” between the implanted scaffold and host tissue to allow tissue remodeling and healing [21]. An ECM composed of collagen, elastin, and native biochemical cues like growth factors, proteoglycans, and hyaluronic acid that promotes tissue regeneration, while the intact natural matrix enables anisotropic load-bearing applications in humans.

An ideal acellular matrix for tissue repair should be biologic, sterile, non-inflammatory, and have sufficient suture retention strength. The versatile properties of the acellular dermal matrix make it a need of the hour in various surgical avenues. Currently, available options for soft tissue defect reconstruction include split-thickness skin grafts, local skin flap coverage techniques, free tissue transfer, autografts, and xenografts [4]. Each has its own set of disadvantages like donor site morbidity, risk of flap/graft complication, and graft rejection. Some defects cannot rely on autografts at all. Acellular matrices are derived from various biocompatible sources including but not limited to human skin, bovine or porcine intestinal sub-mucosal membrane, and urinary bladder. There are a variety of acellular dermal graft products available and each has its own proprietary way of processing, sterilization, and preservation. AlloDerm®, AlloMax®, DermACELL®, FlexHD®, Cortiva®, Dermis on Demand® and GraftJacket™ are current available acellular human dermal matrices while numerous xenografts from bovine or porcine origin include Integra®, MatriDerm®, SurgiMend®, Strattice®, Permacol™, CollaMend®. Acellular dermal matrix graft products also differ in their source, size, thickness, and availability across the globe.

HADs have been extensively used in reconstructive orthopedic surgeries for tendon augmentation procedures where the allograft is thought to get "incorporated into the repair" over time and thus can strengthen the suture-tendon interface. Acellular dermal allografts have been extensively used in reconstructive orthopedic surgeries for tendon augmentation procedures predominantly in Achilles tendon augmentations [22]. Owing to its high suture retention and enhanced healing, HADs are widely used in reconstructing arthritic hands, interpositional ankle arthroplasty, reverse sural adipofascial flap (RSAF) with delayed split-thickness skin graft, glenoid resurfacing, acromioclavicular joint reconstruction, capsular reconstruction to create stability in hips, shoulder and elbow joints [4]. The use of acellular dermal grafts in large or massive rotator cuff tears has shown successful patient-reported outcomes with favorable structural healing rates and decreased progression to arthropathy [23, 24]. The crucial aspect of soft tissue biomaterial graft is the elimination of cellular components with minimal adverse impact on the matrix ultrastructure. To effectively remove nuclear and cellular debris, a combination of hypertonic treatment with detergent treatment was used. Hypertonic solutions create an osmotic gradient that drives the epidermal cells to shrink and disassociate from the dermis. Hypertonic solution facilitates cytolysis and therefore is also an excellent means of decellularization. Coupling with ionic detergents leads to cell lysis and reduction in bioburden by solubilizing the peptidoglycan cell wall, and phospholipid cell membrane and disrupting the nuclear material [8]. This study showed one decellularization method to be highly effective as confirmed by H & E staining. Novel preservation through glycerol enables long-term storage of grafts in hydrated conditions preserving the strength while reducing the pre-op graft preparation time in surgical applications. Freeze-drying serves as an excellent means of preservation in hard tissues; however, the present study confirms that freeze-drying distinctly affects the collagen ultrastructure and mechanical properties of the graft. An ideal process should not affect the composition nor the mechanical properties of the graft, to mimic the native biological tissue. But decellularization and terminal sterilization are of prime importance in the preparation of allografts, therefore minimal alterations in the properties due to the processing steps are inevitable. Biomechanical studies showed sufficient ultimate load to failure capacity (210 N) and suture pull-out strength (126 N) of gly-HAD graft. The recommended use of the graft is in its bilayer folded form at the site of repair which will contribute to its reinforcement.

One issue that precludes widespread use of the allograft is the cost. Most of the allografts currently used in most developing countries, including India, are imported and have limited universal availability. The graft processing technique described in the paper was done in an accredited centre recognized by ROTTO–SOTTO. The laws governing human tissue donation ensure that the human tissue cannot be sold commercially and only reasonable service and processing charges are applied to the product. The product, once marketed, will likely be available at a fair and reasonable cost that ensures universal use. Alternate strategies tried the use of the amniotic membrane, porcine graft and chitosan based artificial grafts but obtained limited success and acceptability. One of the leading products in the segment marketed by Smith Nephew, called REGENETEN,◊ has already been proven to have beneficial results in a study by Bunshell et al. [24]. They showed that the use of this Bovine Collagen tissue decreased stiffness and had bioinductive properties as proven in histopathological studies. As the experience in the arena of orthobiologics have shown, the HAD described here by being a virtue of human derived tissue may be superior alternative however a randomized control study comparing immunogenicity, bio induction and strength may be the way forward.

It is noteworthy that the derived process can be extrapolated to different human tissues including but not limited to blood vessels, pericardium, heart valves, tendons, and ligaments taking into account tissue-specific nuances. Our study employed an effective decellularization method however further investigations on residual DNA content in the processed grafts are required to establish the decellularization criteria of < 50 ng dsDNA per mg ECM dry weight [8]. The human-derived acellular dermis is less thick as compared to a xenograft; this ultimately reflects on the strength of the graft. Although thick in nature, bovine/porcine scaffolds or intestinal submucosal grafts are characterized by a potential risk of rejection [25]. Exploring mechanical reinforcement options of human dermis with biocompatible microporous polymer is the way forward to developing advanced allografts aiming at increased strength. The prospects to develop an ideal soft tissue reconstruction graft choice would be to modify this passive acellular dermal graft into a bioactive graft by seeding with bone-marrow-derived mesenchymal stem cells and/or coupling with biological aspirates and bioactive agents.

Conclusion

A stringent screening criterion with the novel process of chemical decellularization coupled with glycerol preservation ensured minimal adverse impact on the acellular dermal graft native properties. Preserved ultrastructure, sufficient suture pull-out strength, and off-the-shelf availability with minimal risk of disease transmission and rejection make gly-HAD a suitable and affordable graft choice for better surgical reconstruction practices.

Author Contributions

Damini Shah: conceptualization, formal analysis, investigation, methodology, resources, validation, visualization, writing—original draft. Madhu Rathod: resources. Anjali Tiwari: writing—review and editing. Abhishek Kini: project administration, supervision, resources. Prasad Bhagunde: conceptualization, supervision, resources. Vaibhav Bagaria: conceptualization, writing—review and editing.

Data availability

Not applicable.

Declarations

Conflict of interest

DS, MR, and AT are employees of Novo Tissue Bank and Research Centre Pvt. Ltd.

Ethical Standard Statement

This article does not contain any studies with human or animal subjects performed by the any of the authors.

Human and Animal Rights

This article does not contain studies with human participants or animals by any of the authors. Human donated tissues were made available by the Novo Tissue Bank and Research Centre Pvt. Ltd. Release of tissues for improving in house processes and technique was in accordance with family informed consent.

Informed Consent

For this type of study informed consent is not required.