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Point-Counterpoint: Are Living Cells Vital For Achieving Wound Healing In Patients With Diabetes?
Yes.
This author argues that living cells can be helpful in reviving disrupted mechanisms of wound healing and current research suggests the viability of advanced cellular therapy in healing chronic diabetic foot ulcers.
By Alexander Reyzelman, DPM
Due to the epidemic nature of diabetes worldwide, diabetic foot ulcers represent a very common complication of the disease and an ongoing treatment challenge for podiatrists. Podiatrists have the task of identifying at-risk patients in an attempt to prevent an ulcer from occurring, treating an ulcer, reducing the risk of lower limb amputation and mitigating the risk factors associated with recurrence of a healed ulcer.
For the purposes of this discussion, let us assume that podiatrists are appropriately following the accepted algorithm of treating diabetic foot ulcers prior to considering advanced wound care modalities.1 Despite following the standard guidelines such as offloading, management of infection, referral for revascularization when appropriate, debridement and local wound care, we continue to encounter many diabetic foot ulcers that fail to show signs of healing. We frequently refer to these ulcers as stalled and/or chronic.
The question that we often face is which of the many available advanced tissue products we should be using. This is a difficult if not impossible question to answer due to the lack of head-to-head prospective randomized clinical trials available in this arena.
Advanced wound care targets the complex wound healing process of interdependent and overlapping stages. There are various cell types involved in these stages including but not limited to: immune cells, endothelial cells, mesenchymal stem cells, keratinocytes and fibroblasts.2 The delay in diabetic wound healing is a result of defects at a single or multiple stages of the wound healing cascade. The proposed mechanisms responsible for a stalled wound include all of the following: diminished keratinocyte and fibroblast migration, proliferation and differentiation, apoptosis, excessive proteinase and reduced neovascularization.2
Where Advanced Cellular Therapies Can Have An Impact
The rationale for using advanced cellular therapy in chronic wound management is to provide viable cells to the wound bed. Advanced cellular therapy may provide fibroblasts, epithelial cells, mesenchymal cells, a structural matrix and growth factors. It is important to understand that the living cells are able to communicate and respond to the various signals of the wound bed in an effort to supplement the disrupted mechanism of healing.
Research has highlighted the fundamental mechanisms essential to wound healing. At the cellular level, there are multiple causes of a stalled wound. One key process in wound healing is the regulated destruction of proteins by proteinases.3 In chronic, non-healing wounds, this process becomes excessive and counterproductive. Second, increased levels of pro-inflammatory cytokines such as TNF-alpha and interleukin-6 theoretically play a major role in stalled wounds.2 These increased levels of pro-inflammatory cytokines also lead to a prolonged infiltration of leukocytes.2
Mesenchymal stem cells have been the subject of considerable study during the last several years. The role of mesenchymal stem cells in wound healing is not to differentiate into other cell types. The current thought is that mesenchymal stem cells help regulate the wound healing cascade via paracrine signaling.4 These specialized cells assist wound healing through various pathways of inflammation, proliferation of cells and inhibition of detrimental cells.
Keep in mind that the mesenchymal stem cell count within the body declines proportionate to age. Furthermore, mesenchymal stem cells can attenuate inflammation by reprogramming native tissue cells to favor tissue regeneration rather than fibrotic tissue deposit.5 Therefore, the application of mesenchymal stem cells locally to the stalled wound increases re-epithelialization and angiogenesis through upregulation of the expression of growth factors, the downregulation of pro-inflammatory cytokines and the promotion of proliferation of fibroblasts and keratinocytes.2 Keratinocytes are a major source of growth factors that stimulate fibrogenesis and angiogenesis.
At the ulcer’s edge, there is a reduced expression of migration markers.2 The reduction of migration markers reduces the amount of growth factors that signal for wound repair. Fibroblasts are the primary source of extracellular matrix proteins such as collagen and fibronectin. In patients with diabetes, fibroblasts have decreased migration and increased apoptosis. This suppresses the level of extracellular matrix produced, contributing to slower wound contraction.
We also know that impaired angiogenesis is a major contributing factor in stalled diabetic foot ulcers. The cascade of angiogenesis involves growth factors binding to their receptors on endothelial cells of existing vessels. Activated endothelial cells proliferate and migrate into the impaired tissue to form small tubular canals that subsequently mature.
Balancing these various factors that contribute to inflammation may be the holy grail of successful wound healing. In a recent study, Duan-Arnold and colleagues showed that maintaining viable endogenous cells has higher anti-inflammatory activity in comparison to tissues with non-viable cells.6
What The Research Reveals About Living Cell Products And DFUs
Evidence-based research is the ideal platform to utilize when making medical decisions. Although there are a limited number of RCTs involving advanced tissue therapies and no significant head-to-head comparisons between cellular and acellular therapies, there are RCTs that demonstrate the clinical healing success of cellular therapies for DFUs.
Apligraf (Organogenesis) and Dermagraft (Organogenesis) are two living cell therapies approved by the FDA for multiple indications. Most specifically, the pivotal trial with Apligraf conducted in 2009 showed that 51.5 percent of 33 patients healed versus 26.3 percent of 39 patients in the control group.7 Thirty percent of 130 patients in the Dermagraft treatment group showed healing in a randomized controlled trial in comparison to 18.3 percent of 115 patients in the control group.8 Most recently, a multicenter, randomized, controlled trial demonstrated that the living cell therapy of Grafix (Osiris Therapeutics) healed 62 percent of 50 patients with chronic DFUs versus 21 percent of 47 patients treated with standard of care.9
The results of these three clinical RCTs validate the aforementioned basic science concepts. The trial data support the need for living cell therapy to stimulate re-epithelialization in a stalled wound bed.
In Conclusion
Although there are many factors influencing wound healing, there is an abundance of basic science and clinical evidence that support the notion that advanced living cellular tissue therapies are effective in treating chronic, non-healing diabetic foot ulcers.
Dr. Reyzelman is an Associate Professor in the Department of Medicine at California School of Podiatric Medicine at Samuel Merritt University in Oakland, Calif. He is the Co-Director of the San Francisco Center for Limb Preservation at the University of California.
References
1. Kirsner RS, Warriner R, Michela M, Stasik L, Freeman K. Advanced biological therapies for diabetic foot ulcers. Arch Dermatol. 2010;146(8):857-862.
2. Xu, Fanxing, Chenying Zhang, and Dana T. Graves. Abnormal cell responses and role of TNF-alpha in impaired diabetic wound healing. Biomed Res Int. 2013; 2013:745802. Epub Jan. 20.
3. Gibson DJ, Schultz GS. Molecular wound assessments: matrix metalloproteinases. Adv Wound Care. 2013; 2(1):18-23.
4. Maxson S, Lopez EA, Yoo D. Danilkovitch-Miagkova A, Leroux MA. Concise review: role of mesenchymal stem cells in wound repair. Stem Cells Transl Med. 2012; 1(2):142-149.
5. Jackson WM, Nesti LJ, Tuan RS. Mesenchymal stem cell therapy for attenuation of scar formation during wound healing. Stem Cell Res Ther. 2012; 3(3):20.
6. Duan-Arnold Y, Gyurdieva A, Johnson A, Uveges TE, Jacobstein DA, Danilkovitch A. Retention of endogenous viable cells enhances the anti-inflammatory activity of cryopreserved amnion. Adv Wound Care. 2015; 4(9):523-533.
7. Edmonds M. Apligraf in the treatment of neuropathic diabetic foot ulcers. Int J Lower Extrem Wounds. 2009; 8(1):11-18.
8. 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.
9. Lavery LA, Fulmer J, Shebetka KA, Regulski M, Vayser D, Fried D, Nadarajah J. The efficacy and safety of Grafix® for the treatment of chronic diabetic foot ulcers: results of a multi‐centre, controlled, randomised, blinded, clinical trial. Int Wound J. 2014; 11(5):554-560.
No.
These authors say the direct application of cellular therapy has been disappointing and that the emergence of non-cellular modalities that can recruit native stem cells appears to have more promise in healing diabetic foot ulcers.
By Charles M. Zelen, DPM, FACFAS, and William W. Li, MD
The well characterized process of tissue healing involves several important domains of activity:1
• synthesis and structural remodeling of extracellular scaffolding;
• active cellular participation through cell migration, cell differentiation and cellular activation to achieve reparative remodeling and cytokine secretion; and
• hormonal or growth factor modulation of the process
Accordingly, healing in vivo is a complex dynamic that involves all three elements in a process Schultz and colleagues described as “dynamic reciprocity,” in which various components in the process self-modulate the creation of new tissue.1
That an architectural scaffolding made from collagen or similar materials alone is sufficient to promote healing without cells is well known.2 Researchers have shown that collagen itself can facilitate healing and there are a number of collagen only or collagen infrastructure products available.2 These products include Fibracol (Systagenix/Acelity), Promogran Prisma (Systagenix/Acelity), CellerateRX (Wound Care Innovations) and others.
Biological tissues, which are decellularized to remove potentially antigenic material, essentially take the same approach in providing an architecture upon which healing can occur. Other commercially available options of decellularized tissue offer an architecture-only material made from porcine or bovine tissues with any potential antigenic material removed (such as xenografts). Direct application of cellular material in either case is unnecessary.
However, in vivo, native cells are naturally very active participants in healing. Recruited by growth factors and cytokines in the wound, various cells migrate to the area, proliferate and differentiate into the cells needed to lay down new scaffolding, phagocytize damaged tissue, and create blood vessels. Cells such as fibroblasts lay down new collagen and organize an interstitial architecture, resulting in new tissue strength at the site. From both local mesenchymal locations and hematogenous sources, stem cells orchestrate the complex multicomponent system needed for repair. Native cells also secrete additional growth factors to further accelerate the healing process in a balanced way.
Why Cellular Therapy Has Been Underwhelming
Given the importance of cells in this process, clinicians frequently devote effort to delivering live cells, particularly stem cells, to areas where healing is desired. Obvious sources include bone marrow cells, adipose-derived stem cells and others. There are various protocols for placement of the isolated cells into the needed locations. Stem cell isolates are prepared in a number of creative ways and researchers have assessed the use of these modalities via direct injection, infusion into tissues, integration into synthetic materials and adjunctively with other therapies. The results have been somewhat disappointing for several reasons.3-5
First, live stem cells are notoriously fragile, surviving only hours or a day or so outside their normal environment, making isolation and use impractical. Survival decay rates are high, resulting in the application of only a small number of surviving cells to a site — even if one uses the cells immediately — limiting their effect. Secondly, with the exogenous delivery of stem cells, there is difficulty with engraftment and survival in the target tissues with evidence suggesting that they die quickly in vivo. Volarevic and colleagues note, “There are several problems that limit the therapeutic use of MSCs (mesenchymal stem cells) at present. Poor engraftment and limited differentiation under in vivo conditions are major obstacles for efficient therapeutic use of MSCs.”3 These two issues have plagued researchers trying to develop methods for incorporating these useful cells in clinical applications.4,5
Is the direct use of cellular therapy necessary? Early entrants into the field, including Dermagraft (Organogenesis) and Apligraf (Organogenesis), delivered specially cultured cells on a synthetic matrix to a wound, and were an improvement over previous non-cellular approaches. Whether these types of live cells are critical for healing in patients with diabetes has been put to rest with the recent demonstration that the results with EpiFix (dehydrated human amnion chorion membrane) (MiMedx Group) without live cells surpassed the results with Apligraf in a randomized clinical trial.6
Rethinking The Delivery Of Growth Factors And The Recruitment Of Circulating Stem Cells
Modulation of the healing process by growth factors is a necessary component of the healing process and physicians have appreciated this for decades. Indeed, the era of advanced wound care started in the late 1990s with the development of recombinant growth factor PDGF (Regranex, Smith and Nephew). Regranex uses no live cells but does promote healing. While single factor growth factor products in theory are useful and are commercially available, their clinical use has been disappointing.
However, the delivery of a construct of balanced growth factors has shown impressive results when delivered in the balanced milieu of tissue containing their optimal concentrations.7 In nature, it is the balanced action of the various growth factors secreted into the wound environment that orchestrates the simultaneous tissue processes of remodeling and synthesis that characterize normal healing. A good example of such a naturally balanced technology is amniotic membrane, specifically dual amnion/chorion membrane, application to wounds. The results in diabetic wound care have been striking.6
Contemporary research seems to be pointing to a new way of thinking about delivery of all of these components to a wound. The use of extracellular architecture and/or growth factors alone as stimuli to healing through the recruitment of mesenchymal and circulating stem cells appears to be very effective.7 Cytokines and chemokines are enormously potent activators of cellular recruitment to a localized site of inflammation. Using these cell-derived components to attract the patient’s own stem cells may currently provide the most effective way to reboot the patient’s own natural healing ability.
This effect is profound. Recent experiments using a validated model have shown that one can recruit stem cells from the marrow that are concentrated in sufficient amounts to promote healing.8 The growth factors alone may have significant stem cell biological effects.9 This effect is present even in patients with diabetes, in whom stem cells are thought to be less potent. Massee and colleagues have demonstrated that dehydrated human amnion/chorion membrane stimulates Type I and Type II adipose-derived stem cells, increasing their proliferation, and migration, and altering cytokine secretion.10
Three studies have demonstrated that some preparations of amniotic tissues (EpiFix and AmnioFix, MiMedx Group) retain over 50 regulatory proteins including cytokines, chemokines and growth factors present in fresh amniotic membrane.6,11,12 Purion processed amniotic tissues retain the biological activity that directly causes human dermal fibroblasts to proliferate in vitro, upregulating biosynthesis of important wound healing growth factors and causing mesenchymal stem cell migration toward the allograft and wound. Koob and colleagues recently established that these allografts contain angiogenic factors that act as mitogens, inducing microvascular endothelial cells to proliferate and produce more angiogenic factors, and cause endothelial cell migration.13
In Conclusion
Are living cells vital for achieving wound healing in patients with diabetes? Altogether, there has been great debate about whether one needs cells or not to help heal wounds. Clearly, you do need these cells but whether the source of these cells must necessarily be external remains in doubt given the low rates of survival and uptake with this approach. It may be far easier and more effective simply to call cells in from the circulation in an intact host, using the documented impressive ability of growth factors, which are easier to isolate and apply, and can do this alone.14 In clinical randomized controlled studies, this approach is clearly effective in patients with diabetes.11,12
In the future, it is likely that therapies will involve a whole complement of modalities that include cells, architectural proteins, growth factors, and even genetic and epigenetic modifiers. However, the presence of applied living cells, based on the current data, is clearly not a requirement. Applying a balanced natural mixture of growth factors is currently the superior choice.
Dr. Zelen is in private practice at Foot and Ankle Associates of Southwestern Virginia. He is the Medical Director of the Professional Education and Research Institute, Inc. Dr. Zelen is a Fellow of the American College of Foot and Ankle Surgeons. He has disclosed that research funds have been paid by MiMedx Group to the Professional Education and Research Institute to conduct clinical trials.
Dr. Li is the President and Medical Director of the Angiogenesis Foundation in Cambridge, Mass.
References
1. Schultz GS, Davidson JM, Kirsner RS, Bornstein P, Herman IM. Dynamic reciprocity in the wound microenvironment. Wound Repair Regen. 2011; 19(2):134-48.
2. Eweida AM, Marei MK. Naturally occurring extracellular matrix scaffolds for dermal regeneration: do they really need cells? Biomed Res Int. 2015; epub 2015 Oct
3. Volarevic V, Arsenijevic N, Likic ML, Stojkovic M. Concise Review: Mesenchymal stem cell treatment of the complications of diabetes mellitus. Stem Cell. 2011;29(1):5‐10.
4. Wu KH, Mo XM, Han ZC, Zhou B. Stem cell engraftment and survival in the ischemic heart. Ann Thorac Surg. 2011;92(5):1917‐25.
5. Hocking AM, Gibran NS. Mesenchymal stem cells: paracrine signaling and differentiation during cutaneous wound repair. Exp Cell Res. 2010;316(14):2213‐19.
6. Zelen CM, Serena TE, Gould L, et al. Treatment of chronic diabetic lower extremity ulcers with advanced therapies: a prospective, randomised, controlled, multi-centre comparative study examining clinical efficacy and cost. International Wound J. 2015; epub Dec. 23.
7. Koob T, Young C, Lim J, et al. A primer on amniotic membrane regenerative healing. MiMedx/Color House Graphics. Grand Rapids, MI. 2015.
8. Maan Z, Rennert R, Koob T, et al. Cell recruitment by amnion chorion grafts promotes neovascularization. J Surg Res. 2015; 193(2):953-962.
9. Massee M, Chinn K, Lei J, et al. Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro. J Biomed Mater Res B Appl Biomater. 2015; epub Jul 14.
10. Massee M, Chinn K, Lim JJ, et al. Type I and II diabetic adipose-derived stem cells respond in vitro to dehydrated human amnion/chorion membrane allograft treatment by increasing proliferation, migration, and altering cytokine secretion. Adv Wound Care. 2016; 5(2):43-54.
11. Zelen C, Serena T, Denoziere G, Fetterolf D. A prospective randomised comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int Wound J. 2013; 10(5):502-7.
12. Zelen C, Gould L, Serena T, et al. A prospective, randomised, controlled, multi-centre comparative effectiveness study of healing using dehydrated human amnion/chorion membrane allograft, bioengineered skin substitute or standard of care for treatment of chronic lower extremity diabetic ulcers. Int Wound J. 2015; 12(6):724-32.
13. Koob T, Lim J, Massee M, et al. Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration. Vascular Cell. 2014; 6:10.
14. Koob T. Myths of living cell therapies. Document SB266.001. MiMedx Group Inc.
