Decellularization of human dermis using non-denaturing anionic detergent and endonuclease: a review
- 2.3k Downloads
Decellularized human dermis has been used for a number of clinical applications including wound healing, soft tissue reconstruction, and sports medicine procedures. A variety of methods exist to prepare this useful class of biomaterial. Here, we describe a decellularization technology (MatrACELL®) utilizing a non-denaturing anionic detergent, N-Lauroyl sarcosinate, and endonuclease, which was developed to remove potentially immunogenic material while retaining biomechanical properties. Effective decellularization was demonstrated by a residual DNA content of ≤4 ng/mg of wet weight which represented >97 % DNA removal compared to unprocessed dermis. Two millimeter thick MatrACELL processed human acellular dermal matrix (MH-ADM) exhibited average ultimate tensile load to failure of 635.4 ± 199.9 N and average suture retention strength of 134.9 ± 55.1 N. Using an in vivo mouse skin excisional model, MH-ADM was shown to be biocompatible and capable of supporting cellular and vascular in-growth. Finally, clinical studies of MH-ADM in variety of applications suggest it can be an appropriate scaffold for wound healing, soft tissue reconstruction, and soft tissue augmentation.
KeywordsAllograft Homograft Dermis Decellularized Acellular dermal matrix MatrACELL
Decellularization technology has been utilized to remove cellular components in a variety of soft tissues including cardiovascular allograft and human dermal matrix to produce bio-implants for clinical application. The objectives of the decellularization process are to remove potentially immunogenic material and provide a biocompatible scaffold for host cellular and vascular in-growth (Norton and Babensee 2009). Following decellularization, the remaining extracellular matrix can also be used as a scaffold for tissue engineering (Pellegata et al. 2013). Decellularized cardiovascular tissue has been applied in a variety of in vivo applications (Ketchedjian et al. 2005a, b; Hopkins et al. 2009; Elkins et al. 2001a, b; Sievers et al. 2003; Simon et al. 2003; Hawkins et al. 2003; Bechtel et al. 2003, 2005; Kasimir et al. 2006; Steinhoff et al. 2000; Cebotari et al. 2002). Similarly, human acellular dermal matrix (ADM) has been used for wound healing, soft tissue reconstruction, and sports medicine applications. Specifically, human ADM has been reported to be used clinically for repair of rotator cuff tears (Wong et al. 2010; Snyder and Bone 2007; Barber et al. 2008; Burkhead et al. 2007; Bond et al. 2008; Dopirak et al. 2007), during which the dermal matrix is typically used to augment a repair procedure in order to provide biomechanical strength as well as support directed healing. Also, Achilles and quadriceps tendon augmentation procedures using human ADM are reported with satisfying clinical outcomes (Wilkins 2010; Lee 2007, 2008; Barber et al. 2006). In addition, human ADM is commonly used for soft tissue reconstruction procedures including primary, staged, and revision breast reconstruction (Sbitany et al. 2009; Nahabedian 2009; Salzberg 2006) and hernia repair (Kapfer and Keshen 2006; Albo et al. 2006; Candage et al. 2008; Mitchell and Cima 2011). Moreover, human ADM is widely used in the treatment of chronic wounds such as diabetic foot ulcers (Winters et al. 2008; Randall et al. 2008; Brigido et al. 2004).
In particular, one decellularization technology, MatrACELL® (US Patent 6,743,574 (2004)) (LifeNet Health, Virginia Beach, VA), has been applied to human pulmonary patches, which received 510(k) clearance from the FDA and has been in clinical use since 2009 (Lofland et al. 2012). The same MatrACELL technology is also applied to human dermis and the resultant ADM is referred to here as MatrACELL processed human acellular dermal matrix (MH-ADM). The processing, properties, and potential applications of MH-ADM are reviewed herein.
The MatrACELL decellularization and sterilization process
The MatrACELL decellularization process was developed to minimize the impact of processing reagents on biomechanical and biochemical properties of the tissue while still removing cellular components (US Patents 6,734,018 (2004); 7,338,757 (2008)). MatrACELL- processed tissue is rendered acellular in a solution of non-denaturing anionic detergent (N-Lauroyl sarcosinate, NLS), recombinant endonuclease, and antibiotics (including Polymixin B, Vancomycin and Lincomycin). Following decellularization, the tissue is thoroughly rinsed to remove the decellularization reagents. Next, the bio-implant is treated with a water replacing agent, such as glycerol (US Patents 6,293,970 (2001); 6,544,289 (2003); 6,569,200 (2003); 7,063,726 (2006)), prior to final packaging of the tissue. This allows room temperature storage and rapid preparation by the end user. Finally, the bio-implant is terminally sterilized with low temperature, low dose gamma irradiation (Moore et al. 2004). This final step results in a Sterility Assurance Level (SAL) of 1 × 10−6 as anticipated for a medical device, while also inactivating viruses (Moore 2012). The entire process retains biomechanical and biocompatible (Qin et al. 2008) properties of the MH-ADM.
Preclinical evaluation of MH-ADM
MH-ADM was assessed via analytical methods, biomechanical testing, and in vivo analysis. Representative study results are presented in this review. Results from original data sets are not intended to be generalizable, but add novel information to be considered in totality with the other studies presented here.
Histological analysis overview
Analysis of DNA residuals
DNA content for dermis before and after the MatrACELL decellularization process quantified at minimal, nominal, and maximal processing parameters
Average DNA in dermis (ng/mg wet weight)
Avg standard deviation
Average DNA in MH-ADM Dermis (ng/mg wet weight)
Avg standard deviation
Biomechanical testing overview
Moreover, the biomechanical properties of MH-ADM was investigated (Beitzel et al. 2012) in rotator cuff augmentation procedures performed on randomly assigned cadaveric fresh frozen shoulders. Note that MH-ADM is branded as ArthroFLEX® (Arthrex, Inc., Naples, FL) in this publication. The study compared MH-ADM interposed between the bone and tendon as well as placed on top of the repair. Double-row repairs without augmentation served as the control. No significant difference was found in ultimate load to failure between the control group (348.9 ± 98.8 N) and the group with interposed MH-ADM (469.9 ± 148.6 N). However, the group with MH-ADM placed on top had significantly higher load to failure (575.8 ± 22.6 N; P = 0.025) than the non-augmented control (438.9 ± 98.8 N).
Additionally, the biomechanical strength of intact scapholunate ligaments and the ligaments reconstructed with 1.5 and 1.0 mm thick MH-ADM (also described as ArthroFLEX® in this published study by Eshan et al. 2012) was measured in cadaveric tests. While the intact ligament serving as the control failed mid-substance during tensile testing, the 1.0 mm MH-ADM reconstructed ligament failed at the suture-dermal matrix interface and the 1.5 mm MH-ADM reconstructed ligament failed at the suture-bone anchor interface. The authors concluded that the positive results warrant further clinical investigation for using MH-ADM as a potential treatment for chronic scapholunate instability.
Small animal study: in vivo results
Similar results were found for MH-ADM in a study by Capito et al. (2012) where the integrative properties of MH-ADM (also described in the study as DermACELL) and three other ADMs (AlloDerm™, DermaMatrix™ (Synthes, Inc., West Chester, PA), and Integra™ (Integra LifeSciences Corporation, Plainsboro, NJ)) were compared in a rat model. Tissue revascularization, recellularization, and integration were evaluated at four time points ranging from 7 to 42 days. Out of the four ADMs evaluated, MH-ADM had the highest cell density measured at 300, 600, and 900 µm from the blood-vessel graft interface at all time points except Day 42. This difference was statistically significant for many of the time points and distances. Furthermore, MH-ADM had the highest amount of cellular infiltration at all time points, which was significantly greater than two of the other ADMs. Additionally, MH-ADM had a statistically significant greater amount of blood vessel formation in the tissue than the other three ADMs at Day 7 and still had a statistically significant greater amount than two other ADMs at Day 42. In all three objective evaluations, MH-ADM compared very favorably with the other three ADMs tested.
Clinical applications of MH-ADM
Clinical applications of ADMs have been noted in orthopaedic surgeries, dental and craniomaxillofacial repairs, soft tissue reconstruction, and wound healing. Orthopaedic surgeons commonly use ADMs in soft tissue repair procedures to provide additional biomechanical strength and improve healing for rotator cuff repairs, especially for large and massive tears (Wong et al. 2010; Snyder and Bone 2007; Barber et al. 2008; Burkhead et al. 2007; Bond et al. 2008; Dopirak et al. 2007). In addition, ADM was applied to augment Achilles tendon for increased biomechanical strength, possible enhanced healing, and reduced return to activity times (Lee 2007, 2008).
MH-ADM used in tissue replacement of the plantar heel achieved reduced pain involved with ambulation, particularly in weight-bearing areas of the heel (Mulder 2012). In this case series, MH-ADM successfully replaced missing tissue of the plantar heel in 3 patients who had previously lost nearly all of their plantar heel fat pads due to severe motor vehicle accidents. The first patient exhibited encouraging results with pain free ambulation at 6 weeks post-operation and there was continued patient satisfaction at a 3 months post-operative follow-up visit. At over 1 year post-operation, this patient remained pain free. The other two patients did not have long term follow-ups but their initial results were similar to the first patient, supporting the use of MH-ADM for treating plantar defects.
As reviewed here, the MatrACELL process effectively removes cellular material, including DNA and immunogenic components, yielding an acelluar dermis, MH-ADM, which retains biomechanical strength and is biocompatible. Both preclinical and clinical results support the use of this allograft tissue in a myriad of clinical applications, including tendon augmentation, facial reconstruction, wound healing, soft tissue reconstruction, and dental procedures.
- Bechtel JF, Gellissen J, Erasmi AW, Peterson M, Hiob A, Stierle U, Sievers HH (2005) Mid-term findings on echocardiography and computed tomography after RVOT-reconstruction: comparison of decellularized (SynerGraft) and conventional allografts. Eur J Cardiothorac Surg 27:410–415CrossRefPubMedGoogle Scholar
- Chen SG, Tzeng YS, Wang CH (2012) Treatment of severe burn with DermACELL®, an acellular dermal matrix. Int J Burn Trauma 2(2):105–109Google Scholar
- Hawkins JA, Hillman ND, Lambert LM, Jones J, Di Russo GB, Profaizer T, Fuller TC, Minich LL, Williams RV, Shaddy RE (2003) Immunogenicity of decellularized cryopreserved allografts in pediatric cardia surgery: comparison with standard cryopreserved allografts. J Thorac Cardiovasc Surg 126:247–252CrossRefPubMedGoogle Scholar
- Ketchedjian A, Jones AL, Krueger P, Robinson E, Crouch K, Wolfinbarger L Jr, Hopkins R (2005) Recellularization of decellularized allograft scaffolds in ovine great vessel reconstructions. Ann Thorac Surg 79(3):888–896; discussion 896Google Scholar
- Levenda A, Sanders N (2012) Arthroscopic technique for augmentation of rotator cuff with a new acellular dermal matrix. Annual meeting of Arthroscopy Association of North America, Orlando, FLGoogle Scholar
- Moore MA, Jones A, Gaskins B, Wolfinbarger L (2004) Adaptation Of ANSI/AAAMI/ISO 11137 Method 2B sterilization validation for medical devices to tissue banking. American Association of Tissue Banks Annual Meeting, ChicagoGoogle Scholar
- Pellegata AF, Asnaghi MA, Stefani I, Maestroni A, Maestroni S, Dominioni T, Zonta S, Zerbini G, Mantero S (2013) Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering. BioMed Res Int 13:1–8Google Scholar
- Qin X, Cotter A, Chen S, Chen J, Wolfinbarger L (2008) Gamma-irradiated human acellular dermis: a potential treatment for wound and soft tissue defects. Society for American Wound Care. 21st Annual MtgGoogle Scholar
- Steinhoff G, Stock U, Karim N, Mertsching H, Timke A, Meliss RR, Pethig K, Haverich A, Bader A (2000) Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue. Circulation 102(19 Suppl 3):50–55Google Scholar
- US Patent 6,293,970 (2001) Plasticized bone and soft tissue grafts and methods of making and using sameGoogle Scholar
- US Patent 6,544,289 (2003) Plasticized bone grafts, and methods of making and using sameGoogle Scholar
- US Patent 6,569,200 (2003) Plasticized soft tissue grafts, and methods of making and using sameGoogle Scholar
- US Patent 6,734,018 (2004) Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants producedGoogle Scholar
- US Patent 6,743,574 (2004) Process for devitalizing soft-tissue engineered medical implants, and devitalized soft-tissue medical implants producedGoogle Scholar
- US Patent 7,063,726 (2006) Plasticized bone grafts and methods of making and using sameGoogle Scholar
- US Patent 7,338,757 (2008) Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants producedGoogle Scholar
- Vashi C (2014) Clinical Outcomes for breast cancer patients undergoing mastectomy and reconstruction with use of DermACELL®, a new acellular dermal matrix. Plast Surg Int 2014:1–7Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.