A Novel Regenerative Tissue Matrix (RTM) Technology for Connective Tissue Reconstruction

The goal of regenerative medicine is to recapitulate in adult wounded tissue the intrinsic regenerative processes that are involved in normal adult tissue maintenance. Recent advances allow adult wounds to heal in a similar fashion to the regenerative healing that is also present during fetal development. Research suggests that tissue loss or injury that occurs during early fetal development can be corrected by a regenerative mechanism since fetal wound healing appears to occur without scar formation.¹ However, later in the gestational development there is a transition from the regenerative to the reparative healing process, which utilizes fibrosis and scar formation to replace damaged or otherwise wounded connective tissue. Scar does not have the native structure, function, and physiology of the original normal tissue.
When a wound exceeds a critical deficit it requires a scaffold to organize tissue replacement, and 3 pathways or mechanisms of action may exist for the body to respond to the implanted material (Figure 1). A synthetic material or an extracellular matrix (ECM) that has been intentionally crosslinked to avoid enzymatic degradation will elicit a foreign body response towards the implant resulting in encapsulation. Foreign bodies have an increased potential for long-term infection and extrusion of the implant. When a temporary, resorbable synthetic or a poorly processed ECM is employed for wound closure, an inflammatory response will result in resorption of the implant with the deposition of a reparative scar to close the wound. In contrast, a regenerative tissue matrix (RTM), comprising a structurally and biochemically intact ECM implanted at the wound site, supports the appropriate cascade of cellular events characteristic of tissue regeneration ultimately leading to remodeling and transition of the RTM to tissue resembling that which was lost. Therefore, it is critical to understand these differing mechanisms of action in order to understand how the process for preparing the ECM determines which mechanism of action will be utilized by the body.
While regenerative healing is characterized by the restoration of the structure, function, and physiology of damaged or absent tissue, reparative healing is characterized by wound closure through scar formation. Reparative healing begins with the deposition of a provisional protein scaffold of fibrin as a result of hemostasis. Although transitory in nature, this fibrin scaffold serves to organize the healing process through several functional activities.² Initial platelet activation triggers a release of growth factors and other morphogens that become deposited within the fibrin scaffold. In addition to the immobilized growth factors, this scaffold contains cell adhesion proteins that exhibit specific binding to a variety of integrin receptors found on the surface of inflammatory fibroblasts and lymphocytes. These interactions coupled with accommodative protease activity stimulate cell migration into the scaffold. The eventual fibrinolysis and matrix elaboration by the cells within the provisional scaffold along with vascularization of this new connective tissue ultimately results in scar tissue formation. This tissue has a characteristic structure, cellularity, and vascular pattern that are clearly distinguishable from the original, native connective tissue prior to injury. While scar tissue often serves a critical role in the survival of an organism, clinically it is considered a pathological state exhibiting suboptimal functional, biomechanical, and physiological characteristics compared to normal, native connective tissue.
Contrary to the formation of reparative scar, connective tissue structure and physiology are maintained through a process of intrinsic tissue regeneration.³ Connective tissue is responsible for a variety of functions in the adult human and mesenchymal stem cells that are present in the bone marrow, as well as locally within tissues, are believed to enter the circulation in small quantities ultimately localizing within tissues to provide a rapid source of cells for tissue replenishment and regeneration.^4,5 Through the integrin class of cell surface receptors these cells recognize and adhere to the extracellular matrix within a tissue or organ. Once bound to specific adhesion sites within the matrix, they commit to a specialized path of differentiation by responding to local growth factors, morphogens, cytokines, as well as local biomechanical forces. Once cellular differentiation has occurred the cells begin remodeling and restoring the matrix within the tissue.

The ECM in Support of Regenerative Healing

The 3 critical constituents for tissue regeneration are an intact scaffold with the appropriate initial biomechanical properties and the capacity to support the regenerative healing process, the proper binding molecules for growth factors within the scaffold that can support cell differentiation, proliferation, and migration, and lastly, cells capable of responding to growth factors and biomechanical stimuli. As the newly regenerated tissue achieves a mature cellular and vascular status, the resident cells should respond to biological and physical stimuli by remodeling the initial scaffold into a functional, metabolic tissue that performs the function of the original tissue prior to injury.
Processing is critical for the ultimate performance of the scaffold because certain physical and chemical procedures can alter structure and destroy biochemistry, which will lead to a dramatically different mechanism of action upon placement of material in the wound. Presumably, if the scaffold contains the proper structural and biochemical components and is capable of performing as the scaffold in native tissue, the regenerative process should lead to a remodeling and transition to the appropriate tissue with minimal, if any, scarring. The properly prepared ECM must be acellular and therefore not stimulate a foreign body response, which would interfere with the dynamic equilibrium that balances synthesis, degradation, and deposition of new tissue characteristic of regenerative healing.
Regenerative tissue matrices produced using the proprietary LifeCell Technology (LifeCell Corp, Branchburg, NJ), such as GraftJacket^® (Wright Medical Technologies, Memphis, Tenn) and AlloDerm^® (LifeCell Corp, Branchburg, NJ), are created through a process that eliminates cellular materials from human skin, which act as targets of acute immunological rejection. The remaining dermal matrix retains a structurally intact basement membrane, intact collagen fibers, and elastin-rich microfibrils for biomechanical integrity, as well as the biochemical components, such as small, leucine-rich proteoglycans (decorin and biglycan) necessary to foster angiogenesis, cellular migration and the overall intrinsic regeneration process.^6–11
This monograph will review existing literature on the use of GraftJacket RTM in chronic wounds, as well as document several examples of anecdotal human clinical biopsies that indicate that it is possible to replace tissue within a defect through regeneration and transition rather than through reparative scarring.

Evidence for RTM Incorporation

In successful regenerative connective tissue replacement, the RTM becomes repopulated with the patient’s cells, a mature vascular network develops and the tissue becomes so well-integrated into the patient that identification of the new tissue within a clinical biopsy becomes difficult even with employment of specialized histological stains. Since neither scar tissue nor fascia contain significant amounts of elastin microfibrils in a network organization, the Verhoeff von Giesen stain is a valuable tool to identify the location of the RTM in the context of regenerative healing as compared with reparative scarring. This approach has been used previously as a means of studying the integration of RTM into the surrounding tissue.^8,12,13
The difference between an acellular matrix retaining the proper structure and one with documented structural or biochemical damage is readily visualized (Figure 2). Verhoeff’s staining shows RTM with normal, organized architecture, while a human dermal ECM produced by an improper freeze drying process shows destruction of the scaffold integrity.
Clinical biopsies have been obtained from a variety of surgical applications of RTM for tissue regeneration and the samples have been studied using differential staining with hematoxylin and eosin (H&E) and Verhoeff’s staining. Specimens taken by 2 surgeons from different patients at 8- and 12 months have been studied. In the first example, RTM was used to reinforce primary closure of anterior rectus fascia following ventral hernia repair. At 8 months, the patient developed another hernia adjacent to the initial RTM repair that was unrelated to the RTM repair itself. Grossly, the RTM was indistinguishable from the surrounding fascia, but a punch biopsy was taken at the midline of the initial repair through the suspected RTM tissue into the underlying rectus fascia. The tissue was cellular to an apparent cell density for fascia, vascular and well integrated with the surrounding tissue (Figure 3). A distinct tissue plane between RTM and fascia was not discernible. Upon staining for elastin, it was apparent that remnants of the RTM were present and the elastin appeared to be in the process of removal from the starting scaffold. This would be consistent with a dermal scaffold being transitioned into a fascia-like tissue. The collagen in this tissue appeared to remain intact and robust in the face of the process of elastin remodeling.
In the second example, RTM was used in a transverse rectus abdominus musculocutaneous (TRAM) flap reconstruction of a breast following mastectomy. Regenerative tissue matrix was used in the TRAM donor site to reinforce the posterior rectus fascial sheath to minimize the risk of abdominal bulging or hernia formation.¹⁴ At 12 months post reconstruction the patient required an open hysterectomy operation by another surgeon. Upon entry to the abdomen through the previous TRAM donor site, a biopsy of RTM tissue was taken that had the gross appearance of fascia. Examination of the biopsy under low magnification following H&E staining revealed a distinct plane of tissue with a robust collagen matrix, as well as a cellular and vascular density that was consistent with normal fascia and distinct from that of scar tissue. Staining for elastin demonstrated that a significant area of the fascia-like RTM tissue appeared to have been remodeled (Figure 4). Higher magnification allowed the visualization of the robust collagen network and vasculature.

Applications of RTM

Regenerative tissue matrix has been used for a variety of applications including treatment of full-thickness burns,¹⁵ soft-tissue augmentation,¹⁶ and the reconstruction of pelvic, abdominal, and chest walls.^7,17 In particular, the ability to become vascularized equips the RTM with the ability to resist infection, even in the presence of contamination.^18,19
Closure of tissue defects or replacement of lost tissue represents the broadest use of an RTM where the regenerative nature of the scaffold is of paramount importance to its success. Patients exhibiting multiple comorbidities, such as wound site infection, diabetes, obesity, chemotherapy, corticosteroid or radiation therapy, all of whom have a decreased ability to heal properly without complication, represent an added challenge to successful wound treatment.²⁰
Applications of GraftJacket for the successful closure of the diabetic foot have recently been reported.^21–23 Studies highlight the use of GraftJacket in challenging and compromised patients suffering from diabetes. In many of these patients microvascular occlusive disease can cause ischemia and impaired repair.^24,25 Acute complications of diabetes, such as diabetic ketoacidosis,
nonketotic hyperosmolar hyperglycemia, and hypoglycemia are also detrimental to wound healing.²⁶
In a 4-week pilot study of 40 patients, a group treated by sharp debridement followed by a single application of RTM (GraftJacket) was compared with those treated by debridement alone.²¹ At the end of the trial, RTM-treated wounds closed statistically faster than those treated by conventional means. Subsequently, a 16-week prospective, controlled randomized clinical study followed and compared patients in the same way.²² The authors observed 12 out of the 14 patients were healed by week 16 compared to 4 out of the 14 in the control group. Another example of the utility of RTM with this challenging patient group involved a retrospective study of deep wound healing in 17 patients with diabetes. It was reported that 14 of the 17 chronic wounds healed in the 20-week evaluation period with a single application of RTM. The mean wound duration was 8.9 ± 2.7 weeks.²³

Conclusion

The ability to restore structure, function, and physiology is the goal among surgeons of all specialties and may be possible through harnessing the intrinsic regenerative process in adult wound healing and tissue replacement. All biologic scaffolds are not the same because of differences in the methods used to process them—materials that encapsulate and scar do not offer the benefits of regenerative healing but lead to suboptimal results. An RTM that is prepared properly to preserve required elements of structure and biochemistry provides a valuable resource to the clinician and enables the patient to heal by a regenerative process rather than reparative one.