Novel Stabilization and Sterilization Method for Collagen-based Biologic Wound Dressings

Chronic wounds with a variety of etiologies represent a longstanding and major healthcare problem. Among these, diabetic ulcers are a growing and difficult medical challenge with more than 800,000 annual diagnoses in the United States alone.¹ Lack of an ideal treatment modality has wide ranging implications including quality of life (amputations to death) and economic issues. A variety of different approaches have been attempted at addressing medical solutions for chronic wounds, such as generic dressings, peptides, growth factors, and live cellular grafts that report mixed results.^2–6 This is due in part to the multitude of comorbidities associated with chronic wounds, all of which play definite roles in eventual healing.

The logical approach to treatment of any disease condition is in understanding the pathophysiology of the disease state, which in turn can help identify appropriate intervention steps. This is particularly true in palliative treatment modalities, such as biologic dressings for chronic wounds, where symptoms and not the underlying problem are primarily targeted. Thus understanding the biologic balance/imbalances of the healing environment will be able to provide the first and best clues to a treatment option.⁷ Indeed, studies predicated at inhibition and/or modulations of biologic markers that are altered in chronic wounds (as opposed to acute wounds) have been published.⁸ However, the success of such treatments could be enhanced by the concomitant presence of an ideal extracellular matrix (ECM) dressing. A variety of biologic ECM-based wound dressings have been used in wound care settings, again with limited clinical success. In a review of these collagen-based materials Ehrenreich and Ruszczak⁶ have identified several important attributes that would ensure the best clinical success of a biologic wound dressing.
An alternative approach to the biologic basis of treating chronic wounds is presented here. Specifically, the approach addresses both the scientific understanding of chronic wounds as well as device attributes to provide an evidence-based option for choosing a biologic wound dressing.

Biologic Environment of Chronic Wounds

While etiologies (diabetic, pressure, arterial, or venous ulcers, etc) of chronic wounds differ, there are similarities in basic factors that result in delayed healing of these wounds. For example, Trengove et al⁸ have shown that there is a clear dichotomy in levels of proteases and their inhibitors between acute and chronic wound environments. Specifically, there is a 50–100 fold increase in the levels of several metalloproteases (MMPs) with a similar concomitant decrease in tissue inhibitors of MMPs (TIMPs) of chronic wounds. Conversely, Yager and Nwomeh⁹ have shown that chronic wounds in general have elevated neutrophil (PMNs) infiltration, which creates an abnormally proteolytic environment of these wounds. Since most chronic wounds have persistent bacterial infections, a robust PMN response is a necessary host response that is also observed. While infections are generally controlled with antibiotics, bacterial by-products (endotoxins) are also known chemoattractants for PMNs, which release a variety of proinflammatory cell and matrix degrading factors in response.¹⁰ Continued degradation of an ECM-based dressing might also exacerbate this inflammatory environment, further delaying healing. Additionally, vascularity, lack of appropriate levels of growth factors, and inappropriate remodeling cells are all reported in chronic wounds.¹¹ Conceivably, these factors are precursors to a local environment that will be nonconducive for a standard biologic dressing to participate in the healing response. An ideal biologic dressing candidate will be able to resist early degradation, deliver agents that can help facilitate the healing response, and also be biocompatible and sterile, providing additional safety from a patient’s perspective.

Tissue Stabilization Process

Stabilization of ECM tissues is one known method to enhance the biological longevity of a collagen-based material.¹² Over the past 50 years a variety of stabilization (“cross-linking”) methods have been described. The commonly used methods include the use of chemicals (ie, glutaraldehyde, isocyanates, sugars, carbodiimides, etc.), mechanical means (eg, heat), and/or radiation (eg, UV, gamma) in the presence of activators.¹³ In general, chemical and heat cross-linking methods tend to stiffen the tissues, as the process entails elimination of water, and tightening of the collagen strands in the treated matrix. This tends to leave these materials as poor substrates for subsequent cellular infiltration and remodeling. In addition, published reports also indicate a number of side effects associated with these methods. Glutaraldehyde-treated tissues, for example, have a propensity for continued cytotoxicity and calcification.^14–15 Denaturation of collagen is a common side effect of heat and radiation cross-linked tissues, which makes them susceptible to serine proteases, such as trypsin.¹⁶ Finally, all conventional cross-linking methods are also uncontrolled processes—there is no control over the degree of cross-linking in the treated tissues, until exposure to the cross linker is terminated.
The UltiFix^® (Pegasus Biologics, Irvine, Calif) stabilization is a water-soluble carbodiimide (EDC)-mediated chemical cross-linking process that produces stable amide bonds and a biocompatible end product.¹⁷ Pegasus Biologics has licensed this proprietary technology to both stabilize and derivatize a variety of collagenous structures. Several unique features distinguish this stabilization method from conventional cross-linking methods:
Unlike conventional cross-linking processes, this methodology allows for control of the degree of cross-links that are introduced in the treated tissue (Figure 1). This allows for the production of biologic implants (“bioimplants”) with defined cross-link densities as evidenced by their differential susceptibility to remodeling enzymes (Figure 2). Differential stabilization is also traditionally confirmed when cross-link levels are measured by thermal denaturation temperatures as well.
Whereas traditional cross-linking methods tend to produce shorter and stiffer cross-links, this stabilization method yields elastic cross-links through the timely introduction of spacer molecules. This results in a cross-linked tissue that is pliable and has a natural feel and texture, as opposed to the stiffer and often leathery appearance of tissues cross-linked using conventional methods.
Unlike most conventional chemical cross-linking agents, the primary chemical (EDC) in this methodology degrades naturally. This distinguishing factor results in a biocompatible, stabilized product without the known side effects, such as chronic inflammation and/or calcification,¹⁷ when the bioimplant is placed in vivo (Figure 3–5).
Finally, through both published literature¹⁸ and internal documented studies, the EDC-stabilization methodology allows attaching secondary molecules (eg, peptides, growth factors, proteoglycans, etc.) of interest to the collagen matrix. This would allow for generating biological wound care matrices that can direct an appropriate and optimized healing response.

Tissue Sterilization Process

Assurance of microbiological safety is one important requisite for any biological device to be used in clinical settings. While a variety of sterilization systems are currently available, they usually present with 1 of 2 (sometimes both) drawbacks. For example, chemical sterilants like ethylene oxide (EtO) or glutaraldehyde are known to elicit a chronic inflammatory response in humans.^14,19,20 In contrast, radiation, a commonly used method to sterilize, can cause matrix alteration (by denaturization) resulting in accelerated tissue break down,¹⁶ and in instances are also reported to generate toxic metabolites (lipid peroxides).²¹ Thus a sterilization system that not only ensures microbiological safety but also avoids tissue damage and biocompatibility issues, such as those mentioned above, has great value.
The UltiSter™ technology (Pegasus Biologics, Irvine, Calif) is a tested liquid chemical (EDC-based) sterilization method that can inactivate all classes of pathogens.²² The sterilization process has been proven to eliminate vegetative, spore formers, as well as all families (DNA/RNA, enveloped/nonenveloped) of viral pathogens. The model pathogens used to test the robustness of the sterilization system includes hard-to-kill pathogens, such as mycobacteria, clostridium, and porcine parvovirus (PPV) (Figure 6). Aided by a combination of methods to facilitate penetration, such as pH, salts, solvents, temperature, mechanical agitation, etc., chemicals in the UltiSter cocktail gain access to microorganisms in the bioimplant. The natural chemistry of these agents causes inactivation of life-sustaining internal proteins of the pathogens, thereby achieving sterilization. Similar to the UltiFix methodology, the sterilization process has several unique features when compared to conventional sterilization methods:
Unlike conventional chemical sterilants, the primary chemicals employed in this sterilization technology naturally break down resulting in water soluble, nontoxic, and biocompatible residues. A gradual increase in the viability of indicator fibroblast cells exposed to the sterilant with negligible cytotoxicity past the quarantine period following sterilization can be demonstrated (Figure 6).
This feature allows tissue-based bioimplants to be terminally sterilized (ie, sterilization of processed implants in the final package). The sterilized tissues are also subsequently stored at room temperature enabling them to be “off-the-shelf” ready for clinical use with minimal handling requirements.
This sterilization technology specifically targets pathogens while sparing collagen in the tissue. This preserves the structural architecture of sterilized tissues, which is invariably compromised in the presence of conventional sterilants, such as gamma irradiation, activated hydrogen peroxide, and sodium hydroxide (Table 1).
Unlike conventional sterilants, the degradation products in the UltiSter system are not potentially inflammatory (bioimplant bound aldehydes in glutaraldehyde sterilized tissues) or continually graft damaging (free O₂ radicals in gamma and peroxide sterilized tissues). This allows for preservation and maintenance of the biocompatibility properties of the sterilized bioimplants (Figure 4).

Functional Properties of Processed Bioimplants

Bioimplants processed using the UltiFix and UltiSter technologies have received marketing clearance (Class II Medical Device) from the US Food and Drug Administration (FDA). The OrthADAPT™ (Pegasus Biologics, Irvine, Calif) product line is cleared for a variety of soft tissue reparative surgeries in orthopedic indications and the Unite™ Biomatrix (Pegasus Biologics, Irvine, Calif) product line as a wound dressing for a variety of chronic wounds including diabetic ulcers. These products are equine pericardia based, which go through a decellularization process prior to the stabilization and sterilization methods described here. The final product is stored at room temperature and ready to use “off-the-self,” retains the native structural attributes of the raw material (Figure 7).During the course of obtaining the FDA approval, the product successfully passed the entire ISO 10993 panel of biocompatibility tests (Table 2). These include demonstration of biocompatibility following long-term implantation (up to 6 months) of the product (Figure 4). Histopathology analyses of these samples show the appearance of new blood vessels and host cell infiltration supporting the compatibility of these stabilized and sterilized tissues.

Clinical Use of the Biomatrix Wound Dressing

To date there has been a limited use of the Unite biomatrix dressing in light of its limited launch status. The biomatrix dressing has been commonly utilized to treat diabetic ulcers and more importantly in instances where other conventional standards of care did not yield satisfactory clinical outcomes. Representative anecdotal reports of clinical use of the product as a wound dressing are depicted in Figures 8 and 9. The post-operative pictures in these cases demonstrate robust granulation and healing of the wound substantiating the value of the biomatrix dressing.

Discussion

Despite the variety of animal and human based wound dressing products currently available in the market, no particular dressing is noted for being the “standard of care.” The underlying causes of chronic wounds vary and multiple factors contribute to delayed healing and it is impossible for any given wound dressing product to address each factor that facilitates healing. In a recent review, Ehrenreich and Ruszczak6 identify some commonly used products, and more importantly discuss the ideal attributes of such a product. Some of the requisite attributes discussed include compatibility, ability to conform to surface irregularities, resistance to fragmentation, storage and handling requirements, microbiological safety, and minimizing nursing care (ie, number of applications for desired end point). The authors believe that the information presented in this manuscript favorably addresses these key attributes.
When analyzing the state of any chronic wound, the central factor to consider is the biological status of the wound bed. While wounds are generally debrided to create a healthy wound bed, the chronicity of these wounds persists due to several altered physiologies at the cellular level. It is known that chronic wounds have an exaggerated inflammatory response (as opposed to what is considered as normal) leading to a persistent proteolytic environment.⁹ Higher levels of MMPs compared to normal skin, have been documented from patients with chronic wounds.²³ At a cellular level, evidence suggests that altered (higher) levels of MMP production by fibroblasts is one of the factors contributing to delayed healing with aging.²⁴ Similarly, the absence of appropriate vascularization is a known problem in most chronic wounds, and cells from an ischemic or hypoxic skin are known to have higher levels of MMPs combined with unstable collagen production, resulting in continued degradation and consequently, skin ulcers.¹¹ Some of the noncollagen degrading proteases are also known to impair anchoring of the remodeling cells entering chronic wounds.²⁵ Thus, a matrix that is resistant to premature degradation can provide a better environment to progress healing.
The choice of ECM stabilization and sterilization modes play a significant role in the early “removal phase” of donor collagen. Processes that can structurally alter the collagen architecture of the biologic implants can conceivably result in accelerated removal of donor collagen (fast resorption), while those that leave residues or by-products can result in an abnormal inflammatory response. Either of these two outcomes can lead to possible failure of the biologic implant function, and subsequent healing. Carbodiimides have a long history of use in stabilizing a variety of different proteins²⁶ and their chemical safety profile makes them an attractive choice to stabilize ECM matrices for biological use. In a full-thickness wound model, Druecke et al²⁷ report the advantages conferred by a collagen matrix stabilized with EDC in the wound healing response. The clinical success of more than 2,000 clinical applications of the OrthADAPT Bioimplant over the past year confirms the compatibility of an EDC-stabilized collagen matrix in humans. The anecdotal chronic wound care cases presented in this manuscript further complement the effectiveness of a wound dressing processed by the carbodiimide chemistries.
The data shown here demonstrate that these processed biologic implants are highly resistant to enzyme digestion, attesting to the superior stabilization technology of the UltiFix process (Table 1). Additionally, the data also show that the UltiFix technology provides for distinct levels of cross-linking (Figure 2). This would enable clinicians to choose a biologic graft suited to the desired surgical procedure outcome. The tissue sterilization process complements the stabilization technology in preserving the material properties of the tissues while providing the safety of a terminally sterilized biologic implant (Table 1). Among the pathogens inactivated by the sterilization technology are those that are known to resist chemical sterilants, such as Mycobacteria (Mycobacterium sp) and ionizing radiation (eg, porcine parvovirus, Parvovirus sp), highlighting the robustness of the system. Furthermore, the biocompatible nature of the end product allows for optimal remodeling as evident through a variety of small animal and functional studies. In ongoing R&D studies this technology has been extended to generate prototypes of next generation wound dressings. These “smart matrices” have the ability to deliver specific factor(s) that can further improve the healing response.

Conclusion

The novel bioimplant stabilization and optimal sterilization technologies described present an alternative, evidence-based wound dressing option. This technology yields a safe and sterile product that is able to retain its structural properties to yield a biocompatible implant that can aid in the care of chronic nonhealing wounds.

Acknowledgement

The Company would like to acknowledge Jean-Marie Girardot, PhD and M. Nadia Girardot, PhD, the inventors of the UltiFix and UltiSter technologies.
UltiFIX is registered trademark of Biomedical Design, Inc.
UltiSTER, OrthADAPT, and Unite are trademarks of Pegasus Biologics, Inc.
UltiFIX^® and UltiSTER™ are technologies licensed from Biomedical Design, Inc.