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What The Research Reveals About Current And Emerging Cellular And Tissue-Based Modalities

The Centers for Disease Control and Prevention (CDC) predicts that by 2050, one out of every three Americans will be diagnosed with diabetes. About 10 to 25 percent of these patients will develop an ulcer in their life span.1 Among the patients with non-healing ulcerations that lead to amputation, the likelihood of a second amputation is 20 percent within the first year and a 50 percent chance of amputation within three years.2 For those who undergo a below-knee amputation, the five-year mortality rate of the patient increases by 68 percent.

Nonetheless, these statistics have become the incentive force behind research in limb preservation and amputation prevention.2 The field of wound care management has evolved from simple collagen dressings to the use of advanced cellular therapies and placental membrane products. With this in mind, let us take a closer look at different emerging cellular and tissue-based modalities in wound care. Knowledge and proper use of these products can aid in limb preservation and decreasing mortality rates secondary to complications of these non-healing ulcerations.

A Review Of Essential Considerations In The Patient Assessment

Failure to perform a thorough history and physical exam can cause more harm to a patient with multiple comorbidities than the ulcer itself. Glycemic control is essential in patients with diabetes. During the initial assessment, one must also consider contributing factors such as hypertension, hyperlipidemia, atherosclerotic heart disease, obesity, renal insufficiency and, most importantly, ischemic or peripheral vascular disease.

When an ulcer does not go through the stages of wound healing (hemostasis, inflammation, proliferation and maturation) in a timely manner, it is at risk of becoming chronic or non-healing. Any underlying cellular or molecular abnormality in the wound bed or phenotypic abnormality in the dermal or epidermal-derived cells that gives rise to low mitogenic cells and low growth factors can predispose the wound to prolonged and excessive inflammation. In addition, upregulation of immune cells and proinflammatory proteases lead to persistent infection, the formation of drug-resistant microbial biofilms, hypoxia and an inability of dermal and epidermal cells to respond readily to reparative stimuli.3

Therefore, clinicians should consider the currently affected phase of wound healing when determining the use of wound care modalities. This can be based either on the deficiency of the necessary growth factors or abundance of inhibiting factors that are hindering proper healing and closure.

Prior to considering any advanced wound care modality, one must perform a thorough surgical or non-surgical debridement of all non-viable and ischemic tissues. Proper debridement enables clinicians to identify the barriers to healing and select the most appropriate wound care product(s).

When And Where To Use Cellular And Tissue-Based Modalities

With overwhelming research in the field of bioengineered skin substitutes, cellular- and tissue-based products, clinicians may apply these modalities in a sterile manner in the operating room (after appropriate debridement) or in the office setting, for instance, for patients who are not optimal surgical candidates.4 In a recent Q&A discussion, Jeffrey Lehrman, DPM, FASPS, noted that he employs the use of cellular- or tissue-based products for any chronic ulceration that is not decreasing in size by 40 percent in venous leg ulcers (VLUs) or by 50 percent in diabetic foot ulcers (DFUs) after four weeks of treatment.4

What You Should Know About Dehydrated Amnion/Chorion Membranes

Dehydrated amnion/chorion membrane (dACM). This modality is reportedly one of the most widely used human cellular- and tissue-based products (HCT/Ps).3 The product, dACM, is essentially a dehydrated placental allograft consisting of amnion, chorion and a spongy layer. Clinicians may use this product on partial and full-thickness acute and chronic non-infected wounds. Dehydrated amnion/chorion membrane has a shelf life of five years, is easily storable at room temperature and acts by increasing specific growth factors, hydrated proteoglycans, type 3 collagen, transforming growth factor-beta 1 (TGFβ1), hyaluronic acid and regenerative proteins.3,5,6,7

In their recent case series involving 50 patients with lower extremity wounds of various etiologies, Caporusso and colleagues assessed the combination of dACM (NuShield, Organogenesis) with standard wound care.3 They noted an overall median time for complete wound closure of 102 days with 56 percent of wounds healed by week 16 and 73 percent of wounds healed by week 24.

Other dACM products include EpiFix® (MiMedx) and Amniofix® (MiMedx) that are composed of single layer epithelial cells, basement membrane and avascular connective tissue matrix, collagen types 1, 3, 4, 5 and 7 with the amniotic membrane composed of extracellular matrix (ECM), containing fibronectin laminins, proteoglycans, epidermal growth factor (EGF), TGF-β, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF).8,9,10,11

Both of these products undergo the PURION® process, which retains the native composition of ECM as well as signaling molecules and preserves their bioactivity.8,12 In a prospective randomized controlled trial for the treatment of diabetic foot ulcers, Zelen and colleagues showed that 77 percent and 92 percent of chronic wounds at four and six weeks, respectively, healed with a biweekly treatment of EpiFix.13

Could Recombinant Human Type 1 Collagen Have An Impact?

Recombinant human type 1 collagen. Another example of a cellular wound care modality, Vergenix FG (CollPlant) is a flowable wound matrix from tobacco plant-purified fibrillated recombinant human collagen. In a single-arm, open-label multicenter trial, Wiser and colleagues assessed the use of Vergenix FG for chronic lower extremity ulcers of various etiologies.14 They cited a median reduction of wound area by 94 percent in four weeks with minimal adverse events.

Additionally, the study authors emphasized that reduction of elastase and matrix metalloproteinase activity with promotion of angiogenesis and fibroblast chemotaxis alludes to the fact that collagen in its native and intact form has superior outcomes to non-purified, animal-derived collagen in comparison to other available non-human cellular- and tissue-based products.15,16,17

Evaluating Cryopreserved And Lyopreserved Placental Membranes

Cryopreserved placental membrane containing viable cells (vCPM). These are extracellular matrix grafts comprised of collagen, growth factors, fibroblasts, mesenchymal stem cells and epithelial cells of native tissue. This option has the added benefit of application on bone and tendon. In a study by Lavery and colleagues, 82 percent of the chronic diabetic ulcers treated with a vCPM (Grafix®, Smith and Nephew) achieved complete closure in 12 weeks and remained closed throughout the 12-week follow-up phase.18

Lyopreserved placental membrane containing viable cells (vLPM). Another form of placental membrane products, vLPM contains growth factors and endogenous neonatal mesenchymal stem cells.19 The difference is, that by going through a lyopreservation process in lieu of cryopreservation, the viable cells are stored at room temperature. This allows for easier weekly applications and a shelf life of 12 months. In their case series, Reyzelman and coworkers demonstrated that 63.3 percent of patients with lower extremity wounds achieved complete wound closure by week 12 (regardless of etiology) with a mean of six applications of vLPM (Grafix PL Prime®, Smith & Nephew).20

Current Considerations With Human Fibroblast-Derived Dermal Substitutes And Fetal Bovine Dermal Scaffolds

Human fibroblast-derived dermal substitutes. This is another class of widely used cryopreserved products. Dermagraft® (Organogenesis), one of the most common products of this class, is a sterile, cryopreserved human fibroblast-derived dermal substitute, which is derived from fibroblasts of neonatal foreskin.21 By delivering collagen-rich living human dermal matrix to a debrided wound bed, subsequent secretion of structural and metabolically active fibroblasts help facilitate the formation of new granulation tissue. This is achieved through the upregulation and activation of the signaling molecules vascular endothelial growth factor (VEGF), PDGF, FGF and insulin-like growth factor (IGF), fibroblasts, endothelial cells, tenocytes and osteoblasts.21,22

Dermagraft has been FDA-approved for use on DFUs as well as VLUs. It is contraindicated in patients with allergies to bovine proteins as well as wounds with exposed bone or tendon. In a study by Marston and colleagues, there was a 63 percent increase in complete wound closure in diabetic lower extremity ulcers treated with Dermagraft by week 12 in comparison to standard wound care therapy. 21

Another product in this class is Apligraf® (Organogenesis), a bilayered skin substitute comprised of bovine type 1 collagen and human fibroblasts with keratinocytes derived from neonatal foreskin.22 It was the first composite tissue analog the Food and Drug Administration (FDA) approved for diabetic lower extremity wounds. In 2000, the FDA approved Apligraf for an additional indication for venous leg ulcers. Apligraf does not contain any antigen-presenting cells such as Langerhans cells, dermal dendritic cells, endothelial cells or melanocytes. It also does not contain inflammatory cells such as leukocytes. The cells (fibroblasts and keratinocytes) that constitute Apligraf do not persist indefinitely and appear to be relatively short-lived in most patients (less than four weeks).23

Although the exact mechanism is unknown, by providing growth factors and cytokines, Apligraf plays a major role in stimulating differentiation and proliferation of cells in an otherwise non-healing DFU or chronic VLU.22 Falanga and colleagues found that treating venous ulcerations with Apligraf resulted in a threefold increase in complete closure of the wounds by week eight in comparison to the control group.24

Fetal bovine dermal scaffolds. These are regenerative collagen matrix products that promote wound healing through increasing neovascularization. They additionally aid in wound closure by either keratinocyte proliferation and reepithelialization, or by creating a suitable wound bed for split-thickness skin grafting.25 The fetal bovine dermal scaffolds are composed of type 1 and 3 collagens. Type 3 collagen (30 percent fibrillar collagen in fetus versus 10 percent in adults) contributes to scarless wound healing due to the inflammatory response resolving in 14 days.25

PriMatrix® (Integra LifeSciences) is a type of fetal bovine dermal substitute approved by the FDA for use on diabetic, venous, surgical, burn and pressure ulcerations with exposed bone and tendon.

In a retrospective study, Strauss and Brietstein assessed the use of PriMatrix for difficult wounds in 49 patients with 58 wounds.26 They noted that a single application of PriMatrix after surgical debridement facilitated reepithelialization in 58.6 percent of the wounds at 12 weeks. Additionally, in a subset of wounds that had exposed tendon and/or bone, Strauss and Brietstein reported reepithelialization in 61.5 percent of the wounds and successful skin grafting in 19.3 percent of the wounds.26

In a similar study by Karr, lower extremity diabetic and venous leg wounds treated with PriMatrix demonstrated faster healing in comparison to a control group treated with Apligraf.27 Therefore, even though at first glance, PriMatrix is more expensive, the author suggests it may be more cost-effective in wound healing in comparison to another human cellular and tissue product due to the shorter healing time.

A Closer Look At Bilayered Cellular Matrices For Wound Healing

Omnigraft (Integra LifeSciences) is an advanced, acellular, bilayer matrix specifically engineered for dermal regeneration. The dermal layer of the product consists of a three-dimensional matrix of bovine collagen type I and chondroitin-6-sulfate. The epidermal layer consists of a temporary silicone polysiloxane for immediate wound coverage and moisture control. Unlike PriMatrix, one cannot use Omnigraft on exposed tendon and capsule. Omnigraft is FDA-approved for use on neuropathic diabetic ulcerations only.28

OrCel (Ortec International Inc.) is an alternative HCT/P, a form of composite graft harvested from neonatal skin, known as bilayered cellular matrix (BCM), consisting of a type 1 porous bovine collagen sponge. This product contains cultured epidermal keratinocytes and dermal fibroblasts that are cultured in two separate layers.29

OrCel has been approved by the FDA for split-thickness donor sites of burn patients and recessive dystrophic epidermolysis bullosa. The mechanism of action is by assisting in cell migration of cytokines and growth factors FGF-1 (bFGF), nerve growth factor (NGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, hepatocyte growth factor (HGF,) keratinocyte growth factor-1 (KGF-1), (FGF-7), macrophage colony-stimulating factor (M-CSF) PDGF-AB, TGF-1, TGF-2, and VEGF. OrCel, similar to Apligraf, does not contain Langerhans cells, melanocytes, macrophages, lymphocytes, blood vessels or hair follicles.29

In a study by Lipkin and colleagues, in a 12-week period, 35 percent of all diabetic neuropathic wounds treated with OrCel achieved complete closure in comparison to the control group treated with standard therapy.29

What About Extracellular Matrix Scaffolds?

Extracellular matrix (ECM) scaffolds. Comprised of structural and functional proteins, extracellular matrix scaffolds act as tissue-specific templates for constructive tissue remodeling.30 Different ECM products vary in molecular composition due to the type of donor organ and processing method. After an ECM is rapidly degraded, it is replaced with site-appropriate host tissue. Also, ECM products possess antibacterial properties against Staphylococcus aureus and Escherichia coli.30

One emerging and effective extracellular matrix tissue is a porcine urinary bladder-derived tissue matrix (UBM). UBM is composed of a naturally-containing non-cross-linked scaffold and numerous collagens. UBM maintainins its epithelial basement membrane and facilitates cell adhesion, migration and proliferation of in-vivo growth patterns when clinicians use it on an otherwise non-healing chronic wound. UBM is the only ECM product that maintains its basement membrane while going through decellularization, lyophilization and disinfection.

In a 2014 study, Lanterri Parcells and colleagues utilized a UBM (MatriStem, ACell, Inc.) on a series of five patients who had failed wound closure with another HCT/P such as Apligraf, Dermagraft and Integra Wound Matrix.30 The authors noted wound closure occurring  between three and 28 weeks depending on the depth and size of the ulcer, regardless of the etiology of the wound.

The porcine urinary bladder-derived tissue matrix also contains collagen, fibronectin, laminin, glycosaminoglycans and growth factors. Once fully resorbed, UBM leaves functional tissue where scar tissue would have formed. In a study by Rao and coworkers, all of the UBM scaffold was degraded in three weeks.31 UBM is contraindicated in third-degree burns and in patients with known sensitivity to porcine.

Is Small Intestinal Submucosa A Viable Option To Facilitate Wound Healing?

Small intestinal submucosa (SIS). This wound matrix stimulates angiogenesis, connective and epithelial tissue growth, and contributes to differentiation, deposition and maturation of extracellular matrix. Biologically-derived extracellular matrix material originates from a variety of tissues such as urinary bladder, intestinal submucosa, pericardium, liver basement membrane, decellularized Achilles tendon and renal capsule.32 However, the most widely used ECM-derived product has been porcine-derived small intestinal submucosa for the treatment of diabetic, venous and pressure-related lower extremity ulcers.

In study by Niezgoda and colleagues, 49 percent of the ulcers treated with porcine-derived SIS wound matrix (Oasis® Wound Matrix, Smith and Nephew) achieved complete wound closure in comparison to 28 percent in the becaplermin (Regranex®, Smith and Nephew) gel-treated group over a course of 12 weeks.33

In Summary

Non-healing chronic wounds with a higher risk of amputation have a major negative impact on a patient’s quality of life in addition to the long-term financial burden shouldered by patients and our health care system. As a result, one cannot over-emphasize the significance of current and emerging wound care products. Of 13.8 million people with diabetes in United States, there is an approximately six billion dollar annual cost for diabetic wounds alone, which increases to 8.5 billion dollars, if such wounds are complicated by infection and/or amputation.1,2

As I discussed above, the advanced catalyzing and stimulation augmenting roles of dermal substitutes and extracellular matrices, and the regenerative properties of growth factors and collagen along with induction and inhibition of angiogenic and pro-inflammatory cells in placental tissues, respectively, have been proven in various wound care studies.

Research on non-healing chronic ulcerations has shown that reduction in wound size at four weeks is a good predictor of complete closure by 12 weeks for DFUs and by 24 weeks for venous leg ulcers.23 By having a strong understanding of the wound’s etiology and the phases of wound healing, when one encounters a chronic wound with stagnation in healing for three to four weeks with standard therapy, he or she can select the most appropriate advanced wound care modality to help heal the wound.

By expediting wound closure and playing a crucial role in limb preservation, advanced wound care modalities may help minimize life-threatening complications, amputations, financial costs and overall mortality rates. 

Dr. Eragi is a recent graduate of the Coney Island Hospital Wound Care and Limb Salvage Fellowship Program in Brooklyn, N.Y. She is currently in practice with the Newport Care Medical Group in Newport Beach, Calif.

By Golta Eragi, DPM, CWSP, FACCWS

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7. Mcquilling JP, Vines JB, Kimmerling KA, Mowry KC. Proteomic comparison of amnion and chorion and evaluations of the effects of processing on placental membranes. Wounds. 2017;29(6):E38-E42.

8. Koob TJ, Rennert R, Zabek N, et al. Biological properties of dehydrated human amnion/chorion composite graft : implications for chronic wound healing. Int Wound J. 2013;10(5):493-500.

9. Maan ZN, Rennert RC, Koob TJ, Januszyk M, Li WW, Gurtner GC. Cell recruitment by amnion chorion grafts promotes neovascularization. J Surg Res. 2015;193(2): 953-962.

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13. Zelen CM, Serena TE, Denoziere G, Fetterolf DE. A prospective randomised comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int Wound J. 2013;10(5):502–507.

14. Wiser I, Tamir E, Kaufman H, et al. A novel recombinant human collagen –based flowable matrix for chronic lower limb wound management: first results of a clinical trial. Wounds. 2019;31(4):103-107.

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17. Horch RE, Debus M, Wagner G, Stark GB. Cultured human keratinocytes on type I collagen membranes to reconstitute the epidermis. Tissue Eng. 2000;6(1):53-67.

18. Lavery LA, Fulmer J, Shebetka KA, et al. The efficacy and safety of Grafix® for the treatment of chronic diabetic ulcers: results of a multi-centre, conrolled, randomised, blinded clinicl trial. Int Wound J. 2014;11 (5)554-560.

19. Frykberg RG, Gibbons GW, Waters JL, Wukich DK, Milstein FC.  A prospective multicentre, open-label, single-arm clinical trial for treatment of chronic complex diabetic foot wounds with exposed tendon and/or bone: positive clinical outcomes for viable cryopreserved human placental membrane. Int Wound J. 2017;14(3):569-577.

20. Reyzelman A, Vartivarian M, Danilkovitch A, Saunders MC.  A prospective, single-center, open-label case series evaluating the clinical outcomes of lyopreserved placental membrane containing viable cells in the treatment of chronic wounds. Wounds. 2019;31(4):97-102.

21. Marston WA. Hanft J. Norwood P. Pollak R. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care. 2003;26(6):1701-1705.

22. Naughton GK. Mansbridge JN. Gentzkow G. A metabolically active human dermal replacement for the treatment of diabetic foot ulcers. Artif Organs. 1997;21(11):1203-1210.

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24. Falanga V, Isaacs C, Paquette D, et al. Wounding of bioengineered skin: cellular and molecular aspects after injury. J Invest Dermatol. 2002;119(3):653–660.

25. Lanteri Parcells A, Karcich J, Granick MS, Marano MA. The use of fetal bovine dermal scaffold (PriMatrix) in the management of the full-thickness hand burns. Eplasty. 2014;14:e36.

26. Strauss NS, Brietstein RJ. Fetal bovine dermal repair scaffold used for the treatment of difficult-to-heal complex wounds. Wounds. 2012;24(11):327–334.

27. Karr JC. Retrospective comparison of diabetic foot ulcer and venous stasis ulcer healing outcomes between a dermal repair scaffold (PriMatrix) and a bilayered living cell therapy (Apligraf). Adv Skin Wound Care. 2011;24(3):119–125.

28. Driver VR, Lavery LA, Reyzelman, AM, et al. A clinical trial of Integra Template for diabetic foot ulcer treatment. Wound Repair Regen. 2015;23(6):891-900.

29. Lipkin S, Chaikof E, Isseroff Z, Silverstein P. Effectiveness of bilayered cellular matrix in healing of neuropathic diabetic foot ulcers: Results of a multicenter pilot trail. Wounds. 2003;15(7):230-236.

30. Lanteri Parcells A, Abernathie B, Datiashvilli R. The use of urinary bladder matrix in the treatment of complicated open wounds. Wounds. 2014; 26(7):189-196.

31. Rao CN, Reddy P, Liu Y, et al. Extracellular matrix-associated serine protease inhibitors (Mr33,000, 31,000, and 27,000) are single-gene products with differential glycosylation: cDNA cloning of the 33-kDa inhibitor reveals its identity to tissue factor pathway inhibitor-2. Arch Biochem Biophys. 1996;335(1):82-92.

32. Hodde JP, Allam R. Small intestinal submucosa wound matrix for chronic wound healing. Wounds. 2007;19(6):157-162.

33. Niezgoda JA, Van Gils CC, Frykberg RG, Hodde JP. Randomized clinical trial comparing OASIS wound matrix to Regranex gel for diabetic ulcers. Adv Skin Wound Care. 2005;18(5):258-266.

Additional References

34. Niknejad H, Peirovi H, Jorjani M, Ahmadiani A, Ghanavi J, Seifalian AM. Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater. 2018;15:88-89.

35. Gosain A, DiPietro LA. Aging and wound healing. World J Surg. 2004; 28(3):321-326.

36. Zuk P, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-218.

37. Sheikh ES, Sheikh ES, Fetterolf DE. Use of dehydrated human amniotic membrane allografts to promote healing in patients with refractory non healing wounds. Int Wound J. 2013;11(6):711-717.

38. Falabella AF, Schachner LA, Valencia IC, Eaglstein WH. The use of tissue-engineered skin (Apligraf) to treat a newborn with epidermolysis bullosa. Arch Dermatol. 1999;135(10):1219–1222.

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