Recent evidence suggests that stem cells derived from bone marrow have the potential to treat many disorders given their plasticity and ability to differentiate into various types of tissues, including skin cells. Stem cells are known to participate in cell migration and proliferation, contributing to the repair and regeneration of injured tissue, as observed in chronic wounds. This retrospective review of patients treated with autologous bone marrow aspirate (BMA) was performed to determine whether the treatment will help heal lower extremity ulcers by expediting wound closure and reducing the risk of lower limb amputation. The applied technique also exemplifies a simple, safe, and effective method of collecting the aspirate and applying it to the wound directly. Eight patients, 32- to 96-years-old, underwent this procedure between January 2010–March 2010. The subjects had previously undergone at least 1 year of conventional wound care treatment without any signs of healing. Wounds were evaluated and measured at each visit. Three subjects showed a decrease in wound size, and three subjects utilized alternative therapies, including biologic dressings or grafts at week 6, 11, and 16 following surgery since no significant improvement was noted. Although topically applied bone marrow derived stem cells may lead to dermal rebuilding and aid in healing, more research is needed to support their use in chronic wounds.
Chronic lower extremity ulcers are a physical and financial burden to the health and economic establishment in the United States and worldwide. Lower extremity wounds occur in 4% to 10% of people with diabetes, with a lifetime risk of up to 25%, and a 20% to 80% recurrence rate. Foot complications account for about 25% of all diabetic admissions with 100,000 lower-limb amputations performed annually.1 The cost of treating an ulcer may range up to $48,000 per year not including the cost of the amputation or any secondary problems. Early detection and appropriate treatment of these ulcers may prevent up to 85% of amputations. Most lower extremity wounds are caused by venous disease, arterial insufficiency, diabetic neuropathy or a combination of these factors.2 Other etiologies may include vasculitis, pressure, dehisced surgical incisions, burns, and trauma. Venous leg ulcers affect up to 1% of the world’s population, account for almost 70% of all chronic leg wounds with an estimated cost to heal of $6449 per year.2–6 Recent evidence suggests that stem cells derived from bone marrow have potential to treat many disorders given their plasticity and ability to differentiate into various types of tissues, including skin cells.7–12
The primary purpose of expediting wound closure in chronic and problematic lower extremity wounds is to reduce the risk of amputation, as well as associated comorbidities including infection and tissue necrosis. Additional care and cost include but are not limited to the administration of antibiotics, temporary off-loading devices (temporary shoes, contact casts, walkers), hospitalization, and surgery. Expediting closure of lower extremity wounds while decreasing associated complications would have a significant impact on quality of life, rate of morbidity and mortality, and cost of care associated with this population.
Several factors contribute to delayed wound healing at the host level including macrovascular disease, hyperglycemia, and increased venous pressure. In chronic wounds, the senescent cells, due to the inhibition of fibroblast proliferation, are unable to divide and become unresponsive to growth factors. Stanley and Osler13 showed that a human venous leg ulcer with more than 15% of senescent cells would be more difficult to heal.
Application of stem cells in the bone marrow aspirate (BMA) locally to the wound bed has shown promising results for treating lower extremity ulcers. These stem cells are easily derived from the patient’s bone marrow, eliminating the risk of transmission of infectious disease transmission with allogeneic products. The aspirates contain two types of stem cells: hematopoietic and mesenchymal. The hematopoietic stem cells differentiate into red and white blood cells, platelets, and macrophages. The mesenchymal stem cells are multipotent and differentiate into multiple cell types involved in tissue repair when placed in the appropriate microenvironment.14
Bone marrow aspirates consist of inflammatory cell progenitors, which have been shown to participate in wound healing, mesenchymal stem cells, which appear to be phynotypically altered and/or senescent in chronic wounds, and multipotent stem cells.15–19 The progenitor cells show great potential in healing chronic wounds due to their unique immunologic properties and regenerative potential. However, questions still remain regarding the clinical mechanism of cell migration and proliferation, and of extracellular matrix deposition and remodeling after application of BMA-derived stem cells. The most significant problem with BMA use to date is the inability to quantify the number of viable stem cells once the BMA is extracted and immediately put on the wound. Cellular infiltration and regeneration in chronic wounds is poorly defined. Cellular senescence and the presence of biofilm in the wound bed are also important considerations, as they create barriers to healing in the chronic wound. When not adequately addressed, the latter factors are known to impede wound closure and may also prevent effective stem cell activity.
Current treatments and traditional approaches to wound closure, including thorough debridement, have had limited success and do not appear to have significantly decreased amputation rates in patients with underlying deficiency of required cellular activity. While debridement is successful in removing inhibitors and barriers to effective wound closure, including senescent cells, nonviable tissue, and bacteria harbored therein, it does not address the inability of cells in select populations to replicate.
This retrospective review was designed to help determine the potential efficacy and use of BMA-derived stem cells in chronic wounds of the lower extremity of multiple etiologies that had failed all other forms of treatment. The goal of the review was to determine if autologous bone marrow stem cell aspirate had the potential to assist non-healing lower extremity ulcers, which had been unresponsive to traditional methods by expediting wound closure and preventing further need for surgical intervention. The limitations, which are well recognized by the authors, are the relatively small population reviewed and etiology, as well as lack of randomization. However, the purpose of this review is similar to a Phase I trial in determining support for a concept versus demonstrating clinical efficacy. It is not the intent of the paper to discredit potential BMA use.
Patient selection. Patients were selected from the University of California San Diego (UCSD) Medical Center, Department of Surgery, Division of Trauma, by the senior authors without regard to race, gender, ethnicity, or economic status. As a limited number of patients underwent BMA therapy in the time frame reviewed, all patients undergoing the procedure were included, unless insufficient follow up information or documentation was available. Individuals undergoing the therapy all had chronic non-healing lower extremity wounds of 1 year or greater duration that had not responded to traditional methods including a combination of off-loading orthopedic shoes, standard dressing applications, enzymatic debridement, skin substitutes, and surgical debridement. All patients underwent a complete history, physical examination, and imaging studies upon initial presentation, as is standard preoperative practice. The risks, benefits, complications, and alternatives to surgery were discussed with each individual prior to the procedure and informed consent was obtained. At the time of treatment, the patients were not enrolled in any other study or currently receiving any experimental drug or device treatment. Although these data were not part of an active study and is retrospective in nature, IRB approval was obtained to review charts for patients who had undergone BMA treatment. All identifying information was carefully withheld. Any patient with gross clinical infection, including cellulitis or osteomyelitis at the ulcer site, gangrenous changes, active Charcot neuroarthropathy, severe anemia, ankle/brachial index < 0.6, serum albumin < 2.5, renal failure with creatinine > 2.5, malignancy of the lower extremity, or pregnant/nursing, were excluded from BMA treatment since these factors are known to influence wound closure and may significantly reduce surgical benefits until addressed appropriately. With the exception of life threatening situations, it is standard practice to control those factors that might result in surgical failure prior to performing any lower extremity procedure.
Evaluation and procedure. Each patient’s clinical chart was reviewed beginning with the initial visit until the last available progress note. Age, BMI, comorbidities, previous treatments/surgeries, wound etiology, and onset date were recorded. Wounds were evaluated on the basis of size/surface area and underlying pathologic processes. Digital photographs were taken at the initial appointment and subsequent postsurgical visits to monitor wound healing progression.
During a procedure where BMA is part of the treatment, basic surgical procedures are not altered. The patients are taken to the operating room and placed under general or monitored anesthesia. An appropriate lower extremity block is also administered following counseling of the patient by the anesthesia team. The ulcers were surgically debrided to ensure a clean base with no eschar or fibrotic tissue. This allows direct contact of bone marrow cells to a viable wound tissue base. Through a lateral hindfoot approach, a trephine was utilized to harvest the marrow from the ipsilateral calcaneal bone. Approximately 3 cc–5 cc of bone marrow aspirate was collected, depending on the ulcer size. The aspirate was then immediately and directly applied to the wound bed so that the entire wound surface was coated with an even layer of the aspirate and then dressed with an equine xenograft (Unite® [Synovis, Irvine, CA]). While all wounds were covered with a xenograft, at this time there is no evidence to suggest one xenograft product is superior to another except for possible qualitative differences such as cross linkage, which may affect a product’s resistance to proteases. Xenografts were chosen as the external wound explant or cover since they may be left intact until wound closure. As all patients had wounds that were covered with a xenograft and historically data is available on the use of xenografts, any differences from previous data on xenografts alone may be attributed to the addition of the BMA alone versus addition of the stem cell component with the BMA. Figures 1–4 illustrate the wound pre- and post-debridement and BMA extraction, and the application and coverage with xenograft of the BMA. The outer nonadherent and gauze dressings over the xenograft were kept intact for approximately 7 days or until their first postoperative visit, which was ± 2 days. Patients were scheduled to be seen at the wound clinic weekly and had wound measurements taken for 12 weeks or until wound closure, whichever occurred first. The rate of wound improvement and/or failure was documented at each visit only if the xenograft was displaced and the wound was visually examined. If no significant decrease in wound size was recorded (at least 0.5 cm after 6 weeks following surgery), then other therapies were considered to prevent further deterioration or infection.
The study consisted of 8 patients with lower extremity wounds secondary to past burns, vasculitic disease, and venous insufficiency, although secondary diagnosis included trauma, lupus, pyoderma gangrenosum, and/or lymphedema. Age, wound etiology, related comorbidities, wound size, treatments utilized, and surgeries performed for each patient are listed in Table 1. Patients were treated with local dressings before surgery, as well as after surgery, if the xenograft became displaced. Three of the eight patients showed a gradual decrease in wound size over the following few months. One of the three patients had a left saphenous vein radiofrequency ablation per vascular recommendations 3 months following the BMA due to her significant varicosities and vascular disease. Two patients showed progressive increase in wound size several months following the procedure. The remaining three patients showed no significant improvement with < 0.5 cm reduction in wound size after 6 weeks; therefore, alternative therapies were utilized. Two patients proceeded with the application of living skin substitutes to aid in wound closure, and one patient had split-thickness skin grafts placed over bilateral venous ulcers in addition to hyperbaric oxygen therapy.
Autologous adult bone marrow-derived stem cells are known to assist with the tissue repair process by secreting large amounts of growth factors and cytokines. They are capable of differentiating into multiple cell types including endothelium, liver, muscle, skin, bone, cartilage, brain, fibroblasts, and keratinocytes.20 Deng et al21 showed that the fluorescent labeled mesenchymal cells in mice gave rise to stem cells in the skin. In 2008, Rogers et al22 injected bone marrow aspirate topically into the wound periphery in three patients with differing etiologies and suggested that this procedure is a useful and safe adjunct to achieve wound closure. These ulcers healed in 47, 50, and 60 days, respectively.22 Badiavas et al23 achieved similar results in three patients who had complete closure of their yearlong ulcers with use of BMA and cultured cells. All healed within 3 months; however, one patient required a bioengineered skin (Apligraf®, Organogenesis, Canton, MA).23 In 2007, Badiavas et al24 conducted another study injecting BMA into the wounds of 4 subjects but only one healed completely. Our patient review showed wound size reduction in only three of the eight patients without any patient’s attaining closure in the 12 weeks postoperative period. Patients were routinely followed for up to 6 months. Given the non-healing nature of these wounds, other products or procedures were utilized after 6 weeks to augment the healing process when no improvement or deterioration was seen. At 6, 11, and 18 weeks after the application of BMA, two patients received living skin substitutes since the wounds appeared to have little to no intrinsic cell activity. Although there is strong evidence that mesenchymal stem cells can assist in wound healing, there are insufficient human studies with an adequate number of subjects to prove its validity and efficacy.
The largest study to date using bone marrow-derived mesenchymal stem cells with or without autologous skin graft was published by Yoshikawa et al25 in 2008. This study included 20 subjects with various nonhealing wounds. The authors reported complete healing in 18 patients and showed regeneration of native tissue by histologic examination. The study supported previous literature that bone marrow-derived stem cells are associated with dermal rebuilding, remodeling, increase in wound vascularity, and reduced fibrosis.25
Falanga et al26 applied up to three applications of autologous culture-expanded mesenchymal stem cells with a fibrin glue system to acute wounds and chronic wounds. The acute wounds secondary to excision of non-melanoma skin cancers healed within 8 weeks. The chronic, year-long lower extremity wounds significantly decreased or healed in 16 to 20 weeks; however, this healing time does not appear to offer an advantage over healing times of other treatments published in the literature. This study showed a strong correlation between the number of mesenchymal stem cells per square centimeter surface area and reduction in ulcer size. The fibrin glue potentially keeps stem cells in the wound base and migrates out gradually as healing progresses.26 Injection of fibrin glue with adipose tissue-derived mesenchymal stem cells into the fistula tract in 25 patients showed healing rate of 71% with recurrence rate of 17.6%.27
The present results suggest that fresh autologous bone marrow aspirate applied topically may help stimulate healing, but may not necessary lead to a significant visible decrease in wound closure within a given time frame compared to other treatment modalities that are currently approved and available on the US and global markets.
The authors are acutely aware of the many limitations of this review. The purpose of this small retrospective study was to examine a technique for stem cell extraction and determine any proof of concept. We acknowledge that this was not possible, as the patients who had complete documentation and follow up were very limited. Furthermore, as the treated patients were not part of any on-going trial at the time of treatment, two different xenograft products were used. Ulcer etiologies and patient medical histories were also not demographically equal, which makes drawing any conclusions from the results difficult. Two different types of xenograft were used. The two products differ in characteristics, which could have had varying effects on outcomes, as non-cross linked products tend to rapidly degenerate and may be less effective in wounds with high levels of inflammatory enzymes. We do believe that the review provides the reader with information on a new technique that warrants further investigation in a larger randomized controlled trial with demographically equal patients with the same wound etiology. We hope to initiate such a study in the near future. Published data on xenografts alone have suggested that correctly applied, flexible cross-linked xenografts are ideal for problematic wounds, even without stem cells, to help close complex wounds.
1. Bern H, Sheehan P, Boulton AJ. Protocol for treatment of diabetic foot ulcer. Am J Surg. 2004;187:1S–10S.
2. Phillips TJ. Chronic cutaneous ulcers: etiology and epidemiology. J Invest Dermatol. 1994;102:38S–41S.
3. Abbade LP, Lastoria S, De Almeida Rollo H, et al. A sociodemographic,clinical study of patients with venous ulcer. Int J Dermatol. 2005;44:989–992.
4. Callam MJ, Ruckley CV, Harper DR,Dale JJ. Chronic ulceration of the leg: extent of the problem and provision of care. Br Med J (Clin Res Ed). 1985;290:1855–1856.
5. Morrell CJ, Walters SJ, Dixon S, et al. Cost effectiveness of community leg ulcer clinics: randomized controlled trial. BMJ. 1998;316(7143):1487–1491.
6. Trent JT, Falabella A, Eaglstein WH, et al. Venous ulcers: pathophysiology and treatment options. Ostomy Wound Manage. 2005;51(5):38–54.
7. Bianco P, Riminucci M, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells: nature biology and potential applications. Stem Cells. 2001;19(3):180–192.
8. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418(6893):31–49.
9. Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. 2001;105(3):369–377.
10. Pittenger MF, Mackay AM, Beck SC, et al. Multi-lineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147.
11. Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human
embryonic stem cells. Proc Natl Acad Sci U S A. 2000;97(21):11307–11312.
12. Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science. 2000;287(5457):1427–1430.
13. Stanley A, Osler T. Senescence and the healing rates of venous ulcers. J Vasc Surg. 2001;33(6):1206–1211.
14. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276(5309):71–74.
15. Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol. 2001;69(4):513–521.
16. Lingen MW. Role of leukocytes and endothelial cells in the development of angiogenesis in inflammation and wound healing. Arch Pathol Lab Med. 2001;125(1):67–71.
17. Mendez MV, Stanley A, Phillips T, Murphy M, Menzoian JO, Park HY. Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence. J Vasc Surg. 1998;28(6);1040–1050.
18. Raffetto JD, Mendez MV, Phillips TJ, Park HY, Menzoian JO. The effect of passage number on fibroblast cellular senescence in patients with chronic venous insufficiency with and without ulcer. Am J Surg. 1999;178(2):107–112.
19. Vande Berg JS, Rudolf R, Holland C, Haywood-Reid PL. Fibroblast senescence in pressure ulcers. Wound Repair Regen. 1998;6(1):38–49.
20. McFarlin K, Gao X, Liu YB, et al. Bone marrow-derived mesenchymal stromal cells accelerate wound healing in the rat. Wound Repair Regen. 2006;14(4):471–478.
21. Deng W, Han Q, Liao L, et al. Engrafted bone marrow-derived flk-(1þ) mesenchymal stem cells regenerate skin tissue. Tissue Eng. 2005;11(1-2):110–119.
22. Rogers LC, Bevilacqua NJ, Armstrong DG. The use of marrow-derived stem cells to accelerate healing in chronic wounds. Int Wound J. 2008;5(1):20–25.
23. Badiavas EV, Falanga V. Treatment of chronic wounds
with bone marrow-derived cells. Arch Dermatol. 2003;139(4):510–516.
24. Badiavas EV, Ford D, Liu P, et al. Long-term bone marrow culture and its clinical potential in chronic wound healing. Wound Repair Regen. 2007;15(6):856–865.
25. Yoshikawa T, Mitsuno H, Nonaka I, et al. Wound therapy by marrow mesenchymal cell transplantation. Plast Reconstr Surg. 2008;121(3):860–877.
26. Falanga V, Iwamoto S, Chartier M, et al. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 2007;13(6):1299–1312.
27. Garcia-Olmo D, Herreros D, Pascual I, et al. Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum. 2009;52(1):79–86.