Adjunctive Bioengineered Bi-layered Cell Therapy (Apligraf) With Excimer Laser Revascularization Improves Wound Healing

Introduction

Critical limb ischemia (CLI) remains poorly characterized in the clinical literature. Therefore, information, knowledge and awareness surrounding the clinical impact of CLI remains obscure. Sparse data exists on the true prevalence of CLI, but in the United States (U.S.), it is estimated at 1% of the > 50-years-of-age population, which is at least double that incidence in the > 70 years age group.¹ Also, the incidence of CLI is expected to increase significantly with our aging population and expected increase in diabetes.¹ There is an even greater paucity of data and lack of understanding regarding the clinical costs of treating CLI and amputation to the patient, family and society. It is estimated that between 220,000–240,000 major and minor lower extremity amputations are performed in the U.S. and Europe yearly for CLI.^1–5 In the U.S., the amputation rate has increased from 19 to 30 per 100,000 persons/year over the last two decades, primarily due to an increase in diabetes and advancing age.^6,7 Despite advances in cardiovascular treatment in patients over 85 years of age, an amputation rate of 140 per 100,000 persons/year has been reported, with a primary amputation (PA) still carrying an excessively high mortality rate of 13–17%.^7–9 In the highest-risk patients, 30-day periprocedural mortality after amputation can range from 4–30%, and morbidity from 20–37%,^8,10 because many end-stage CLI patients will suffer from sepsis, progressive renal insufficiency and other significant medical comorbidities.

CLI: The Problem

The definition of CLI remains somewhat obscure because a diagnosis of CLI does not depend solely on anatomic or angiographic criteria, and therefore requires both subjective and objective components. The clinical entity of CLI is thus subject to a wide range of patient-to-patient variability, including the degree of tissue injury and the status of collateral blood flow. Multiple classification systems and definitions of CLI are available, including: a) European Working Group on CLI Definition with a clinical criteria of persistent ischemic rest pain requiring analgesics for > 2 weeks or ulceration or gangrene of the foot or digits, plus objective criteria of ankle systolic pressure 8 One of every four diabetics will face CLI within their lifetime, and the diabetic is at risk for an amputation 7–40 times greater than a non-diabetic.^5,6 It is estimated that greater than 15 million patients in the US are diabetic.⁵ Successful rehabilitation in patients after below-knee amputation is achieved in less than two-thirds and less than one half after above-knee amputations.^11–14

Overall, 11–14 CLI is both an incurable and bilateral disease. After successful limb salvage (LS), 20–30% will require a second intervention within 18–24 months to maintain ipsilateral LS.^5,8,13,14 Moreover, 30–50% of diabetic amputees will face contralateral CLI and undergo a second leg (contralateral) amputation within 3–5 years of an ipsilateral amputation.^5,6,13 Therefore, a lifetime commitment to surveillance is a prerequisite for the multidisciplinary physician and healthcare provider if long-term LS is to be achieved. It has been estimated that the total cost of treating CLI in the U.S. alone is $10–20 billion/year.^5,7 The annual cost of follow-up long-term care and treatment for an amputee who remains at home has been estimated at $49,000/year, and only $600/year after LS.5,7,11 For many (estimated 15–20%), an amputation ultimately will result in permanent nursing home placement and these professional nursing care costs after amputation in the U.S. are estimated between $70,000–$100,000 per year.^9,10 Johnson et al. attempted to evaluate the costs to the patient and family for home alterations and accommodation for an amputee.¹² Items ranged from $700 for a toilet seat to $25,000 for concrete wheelchair ramps.¹² It is estimated that just a 25% reduction of amputations could save $2.9–3.0 billion yearly in U.S. healthcare costs.^5–7

CLI: The Natural History

Wolfe et al. described the natural history of CLI in a collation of 20 publications on 6,118 patients by stratifying them into a low-risk cohort of 4,089 patients (rest pain only and ankle pressure > 40 mmHg) and a high-risk cohort of 2,029 patients (rest pain and tissue loss with or without ankle pressure 4 Multiple reports have repeatedly documented the poor overall prognosis for the CLI patient with mortality rates 1,8,13 Within one year of the diagnosis of CLI, 25–30% will require a major amputation and another 25–30% will die.^1,8,13 Interestingly, several recent reports have shown significantly improved long-term survival after revascularization and LS as compared to CLI patients following revascularization failure and amputation.^15–17 Statistically significant 5-year survival rates were achieved after LS in the Kalra et al. report, with an overall 5-year survival rate after LS of 60% versus only 26% after an amputation, documenting a significant improvement in mortality with successful LS.¹⁷

The clinical and economic costs to the CLI patient are extremely high, underscoring the need for a characterization of the multidisciplinary treatments required and involved in treating CLI. The impact of CLI will become more evident especially when considering the incidence of diabetes, and CLI is expected to significantly increase yearly to a global epidemic scale. It seems intuitive that there would be a consensus recommendation, protocol, or algorithm in treating the CLI patient, combining and optimizing adjunctive revascularization and wound care strategies, especially in treating the Rutherford Class 5 (minor ischemic wound) and 6 (major ischemic wound) patient, but there is not. Little data exists on wound care recommendations in the CLI patient with severe arterial insufficiency and few reports exist combining revascularization with the novel class of bioengineered skin substitutes in CLI.

A recommendation for optimal revascularization with optimal wound care in treating CLI should be established. Apligraf® (Organogenesis, Inc., Canton, Massachusetts) a novel bioengineered, living tissue bi-layered cell equivalent, has shown statistical significance in improving wound healing (WH) of chronic venous insufficiency (CVI) ulcers and diabetic foot ulcers (DFU).^18,19 Apligraf has been reported to promote WH and stimulate epithelialization by increasing growth factors and cytokines, stimulating angiogenesis, facilitating cell-to-cell vessel reactions, increasing matrix proteins, and providing a physical and biological barrier against wound infection and desiccation.^18–21 Apligraf is currently approved for treatment of DFU and for treatment of partial-thickness and full-thickness skin loss due to CVI ulcers. Apligraf has been reported effective in treating other chronic and acute wounds, including epidermolysis bullosa, thermal injuries, pressure ulcers, surgical excision sites, donor site wounds and keloids.^20–26 Those clinical reports, the reported WH attributes of Apligraf, and our experience in treating CVI and DFU with Apligraf resulted in the adoption of Apligraf as our primary wound treatment in patients with leg and sternal wound complications after CABG,²⁷ and now for optimizing WH when treating patients with CLI.²⁸

Methods. Between January 1, 1999 and August 31, 2004, 68 patients with advanced CLI (Rutherford Class 5-6 with open ischemic wounds with/without established tissue loss) underwent LS revascularization with percutaneous peripheral intervention (PPI) in 60/68 (88.2%), and surgical bypass in 8/68 (11.8%). Rutherford Class 5 implies minor digital ulceration, and Class 6 more advanced ulceration with tissue loss but a salvageable foot. PPI included excimer laser atherectomy (ELA) (Spectranetics Corporation, Colorado Springs, Colorado) in 19/68 (31.6%), sole balloon angioplasty and/or stent in 2/68 (2.9%), and combined PPI (ELA and balloon angioplasty without stent) in 39/68 (57.3%). Therefore, ELA was used for revascularization in > 80% of our CLI cases. Surgical revascularization included distal tibial bypass surgery (TBS) to dorsalis pedis artery (DPA) in 3/8 (37.5%), peroneal artery in 2/8 (25.0%), anterior tibial artery (ATA) 1/8 (12.5%) and posterior tibial artery (PTA) in 2/8 (25.0%). Traditional wound care (TWC) Group A was applied to 29/68 (42.6) of the CLI wounds [Rutherford Class 5 = 21/29 (72.5%) and class 6 = 8/29 (27.5%)]. Early primary wound treatment consisted of sharp debridement of all devitalized tissue and appropriate systemic antibiotic treatment until the wound bed was free of severe active infection. TWC consisted of a rigid protocol using a single home health care team, daily wet-to-dry saline gauze dressing changes, patient and family wound care education, and weekly physician and nurse practitioner wound evaluation. All wound care recommendations and treatments were directed and performed by the vascular surgeons within a strict outpatient wound care protocol. All wounds were evaluated weekly. Wound sizing was accomplished by computerized planimetry of surface acetate wound tracing and recorded by serial photographs.

Primary wound treatment with Apligraf (Group B) occurred in 39/68 (57.4%) with 30/39 (76.9%), and 9/39 (23.1%) being Rutherford Class 5 and 6, respectively. The optimal Apligraf preparation and application technique has been previously described, and was followed in our patient population.²⁹ The original dressing should be left in place for one week, and then a weekly dressing change protocol should be instituted. Achieving Apligraf wound bed adherence is tenuous for the first two weeks, but optimal results will occur if Apligraf adherence remains intact for 3–4 weeks. We have found Mepitel® (MöInlycke Health Care, Newton, Pennsylvania) to be effective in achieving this purpose. The standard dressing is a nonadherent primary dressing covered by sterile Vaseline gauze and an absorbent cotton wrap bolster. It is important to secure the dressing in place, but compression > 30 mm is not recommended.²⁹ A retrospective multivariable analysis was performed including wound size, location, time to WH, 6-month LS rate and wound treatment (TWC versus Apligraf^®). The time to WH was defined as the time to complete epithelialization of the wound requiring no dressing changes.

Results. The time to WH was substantially less in the Apligraf® Group B (18–89 days, mean = 51), as compared to the TWC Group A (31–138 days, mean = 83). The 6-month LS rate for the Apligraf (B) versus TWC (A) groups were also improved and were 36/39 (92.3%) and 23/29 (79.3%), respectively. Repeat Apligraf applications were required in 7/39 (17.9%) cases at 6 weeks. The overall 6-month LS rate for these 68 end-stage CLI patients was 59/68 (86.8%). The 6-month LS rate for the individual revascularization techniques of TBS, ELA, angioplasty/stent and combination revascularizations was 6/8 (75.5%), 17/19 (89.5%), 1/2 (50.0%) and 35/39 (89.7%), respectively. Secondary revascularization procedures were required within 6 months in 8/60 (13.3%) and 2/8 (25.0%) of PPI and TBS cases, respectively.

Case 1. Combined CVI ulcer with CLI (diabetic heel pressure ulcer). A 59-year-old male with Type II diabetes presented with a large left leg CVI ulcer and large heel pressure ulcer treated for over 4 months with primarily betadine. There were no palpable pulses, a left ABI of 0.35 and duplex ultrasound predicated no pedal arterial flow. Peripheral angiography revealed total occlusion of all infrapopliteal arteries with no pedal flow. Percutaneous excimer laser atherectomy (ELA) was performed after using small-sized coronary artery guidewires (0.018 mm) and the laser to traverse a 100% PTA occlusion. The 0.9-mm Excimer laser was then advanced slowly, providing atherectomy to the tarsal branches of the foot. Small 2.0 mm coronary artery angioplasty balloons were used to facilitate revascularization with excellent “straight-line” flow being provided to the foot. On day 2, post-laser revascularization, the black eschar and devitalized tissues were debrided from both the CVI and diabetic heel ulcer, and multiple Apligrafs were applied. A second Apligraf application was applied to both ulcers at 6 weeks and complete WH occurred at 61 days. The patient remains asymptomatic 2 years later.

Case 2. Rutherford Class 6 CLI with wound dehiscence post TBS. A 64-year-old diabetic female presented to an outside facility with severe rest pain, cold right foot and several dry gangrenous digits. Digits 1, 2 and 5 were amputated at that facility, with poor healing at the amputation sites. She was scheduled for below-the-knee amputation (BKA) at the outside facility after multiple failed attempts at PPI and she was not felt to be a surgical candidate because there was no available autogeneous saphenous vein (prior CABG). She was transferred to our Institute where another LS PPI was attempted but was unsuccessful. A DPA target was identified on angiography during the LS PPI attempt. Two autologous donor veins were obtained, and a composite (sewing 2 veins together for added length) femoral to DPA bypass was performed. On post-op day 6, the distal DPA anastomotic site incision became infected and the incision dehisced, exposing the distal anastomotic site. On post-op day 8, the anastomotic site incision and digital wounds were debrided. Forty-eight hours later, multiple Apligrafs were placed on the exposed distal anastomosis and the digital amputation site wounds. A second Apligraf application was placed at both sites 6 weeks later. Complete WH occurred at both sites by day 68. Rapid degranulation tissue formed over the wound dehiscence and exposed graft at only 3 weeks, and the quality and strength of the final wound scar appeared excellent. The patient remains asymptomatic 18 months later. A laser PPI was performed 6 months later for contralateral CLI.

Case 3. Rutherford Class 6 diabetic patient with advanced tissue loss. A 61-year-old diabetic male presented with advanced gangrene of the left foot after amputation of digit #1 at an outside facility, and was scheduled for BKA. Peripheral angiography and CTA at our facility revealed a total occlusion of all infrapopliteal vessels. ELA of the ATA was performed with excellent results, revascularizing the forefoot. The patient underwent wound debridement and amputation of digits #2 and 3 with multiple Apligraf^® applications 48 hours later. There was significant WH at six weeks, total WH at three months and the patient remains asymptomatic at one year post ELA and LS.

Discussion. In Wolfe’s description of 6118 CLI patients, 25% still required major amputation at one year even after successful revascularization, implying that much more than just revascularization is often necessary to achieve long-term LS, especially in the end-stage CLI patient.⁴ Infection, nutrition, microcirculatory impairment, osteomyelitis and impaired WH on the cellular and microcellular level are but a few explanations of how limb loss can still occur with a “palpable pulse.” A 20–25%, 12-month secondary reintervention rate with both TBS and PPI would also imply that the sooner an ischemic wound achieves total WH, the better for long-term LS. This concept does not seem to be emphasized or even discussed when reviewing the CLI, revascularization or wound care literature.

Most still consider TBS the “gold standard” treatment for CLI. A meta-analysis of 4 TBS studies published since 2001 reveals a 5-year LS rate of 65–78.1% in 1,619 CLI patients (68–100% diabetic).^15,17,30,31 In a landmark article, Pomposelli et al. reported a decade experience with TBS in 1,032 CLI patients (92% diabetic) with excellent 5- and 10-year LS rates of 78.1% and 59.8%, respectively.¹⁵ It must be noted that all of these reports were from experienced institutes committed to LS. TBS, however, has significant limitations, including mortality (1.3–6.0%), wound infection (20–30%), severe graft infection (1–1.5%), myocardial infarction (3%), acute — 24-months graft occlusion/stenosis (15–30%), and inadequate or absent venous conduit in 40–50% CLI of cases because use of the saphenous vein is still the most common conduit for CABG (~ 50% of CLI patients will have coronary artery disease).^5,8,13 TBS “probably” still remains the gold standard and is a successful, but complex, non-repeatable procedure that also is not available in every community.

The pioneering ELA work of Professor Giancarlo Biamino has led to an understanding of the unique thrombus and atheroablative properties of pulsed ELA in contrast to earlier (now abandoned) continuous wave thermal lasers, resulting in the ELA now being a viable option in treating CLI.^32,33 The unique properties and energy of the 208 mm wavelength Excimer laser have now been harnessed and found to be safe and effective in treating CLI.³⁴ The laser simply converts atherosclerotic plaque and thrombus (clot) to C02 for immediate absorption, therefore photoablating the arterial blockage and clot. The landmark Laser Angioplasty for Critical Limb Ischemia (LACI) trial represents one of the only organized multi-center trials addressing a true CLI patient population. Even today, this trial has not received the credit it deserves. The LACI trial enrolled 155 CLI limbs with 423 lesions in 15 U.S. and German sites.³⁵ All patients were considered poor or non-surgical candidates with high comorbidities (Rutherford Class 4 = 29%, and 5–6 = 71%). The arteries treated included SFA = 41%, popliteal = 15%, and infrapopliteal = 41%, with ~ 50% requiring multivessel ELA.³⁵ The LACI trial results included:

• Procedural success (PS) = 90%;

• ELA delivery rate = 99%, despite the 8% failed wire crossing in 100% occlusions, therefore the “step-by-step technique” was utilized;

• Adjuvant balloon angioplasty = 96% and stent = 45% overall (SFA = 61%, popliteal = 38%, and infrapopliteal = 16%);

• Straight line flow to the foot = 89%;

• Six-month LS = 93% with very low periprocedural complications (10% overall adverse events at 6-months);

• A low 6-month reintervention rate of 16%, with 2% requiring TBS.³⁵

The LACI trial demonstrated that PPI in CLI can achieve high PS and 6-month LS rates (93%) in very fragile and complex CLI patients who had no other surgical option with very low complications and reinterventions. Similar results have been recently reported in the “Belgium LACI” and the CIS “LACI Equivalent” studies.^36,37

pligraf bi-layered cell therapy treatment for surgical wound complications was first reported by Allie et al., describing a 4-year experience with 45 post-CABG wound complications (30 leg and 15 sternal).²⁷ Apligraf therapy was found to significantly improve time to WH in both sternal and leg wounds, with less time and healthcare resource utilization. The mean time to WH in the Apligraf leg group was 46 days versus 84 days for TWC. Sternal WH occurred at mean 36 days versus 62 days in favor of the Apligraf group. Bioengineered substances have been developed as living skin tissue replacement to potentate WH by increasing growth factors and cytokines, stimulating angiogenesis and increasing matrix proteins, all necessary for proper WH.^20,21

Basic research has demonstrated that wound repair and closure, and dermal regeneration can be improved and hastened with newly developed, bioengineered, living bi-layered cell therapy.^20,21 The combination of optimizing WH with adjuvant Apligraf therapy in conjunction with revascularization for end-stage CLI patients has rarely been reported. Chang et al. reported the only similar series of patients in 1999, in which 31 patients were randomly assigned to moist dressings versus Apligraf in a variety of foot wounds, but these applications were primarily in TBS patients and at much later times after TBS (within 60 days).³⁸ Chang et al. concluded that Apligraf was significantly more effective in achieving WH than moist dressings in the percentage of wounds healed (62% versus 0% at 8 weeks, 86% versus 40% at 12 weeks, p versus 15 weeks, p = 0.0021, rank-sum test).³⁸

The overall concept of an aggressive multidisciplinary approach to achieve limb salvage would seem intuitive but appears to rarely be applied clinically. In a recent report attempting to identify and characterize the clinical pathways for treating CLI, several disturbing patterns were identified. In an analysis of 417 CLI patients from a 2.5 million U.S. reference population treated between 1999–2001, 67% were treated first with a primary amputation without even consideration for revascularization and LS.39 The literature suggests a primary amputation would be appropriate in 40,41 Shockingly, less than one half (49%) of these 417 CLI patients had any objective diagnostic vascular evaluation prior to a primary amputation with the incidence of ABI, angiography, and MRA being 35%, 16% and 1%, respectively.³⁹

The percentage of referrals to clinicians who can provide revascularization in these 417 CLI patients was vascular surgery (21%), interventional cardiology (26%) and radiology (39%).³⁹ The multiple disciplines involved in diagnosing and treating CLI patients must do a better job of educating and communicating with each other, especially with the recent developments of improved novel revascularization and diagnostic techniques. The recently developed multidetector 16–64 channel computed tomography angiography (CTA) is now available and is safe, accurate, simple, fast (12 CTA is rapidly replacing traditional peripheral angiography as the “gold standard” for diagnosing and planning treatment for CLI.⁴² There should be little reason today for not obtaining this novel, noninvasive, outpatient vascular evaluation in the treatment of the CLI patient. Likewise, multiple novel wound care modalities are now available to the CLI patient to optimize WH and LS with revascularization. Again, multidisciplinary education and communication are paramount if we are to achieve optimal LS rates after revascularization. It is these authors’ opinion that aggressive wound care with optimization of WH should be addressed immediately and in conjunction with revascularization in the end-stage Rutherford Class 5–6 CLI patient.

Unfortunately, few data exists regarding the optimization of revascularization and WH in the CLI patient. Apligraf application has provided this optimization for our CLI patients. There are several limitations to this study that deserve mention. This nonrandomized retrospective analysis carries the known biases inherent with this methodology. The relatively small sample size, short follow-up and the subjective and objective evaluation of WH preclude any definitive recommendations at this time. Attempts to minimize biases include the use of computerized planimetry of surface acetate wound tracings for wound sizing and serial photographs, which are accepted objective measurements of wound care and WH. A detailed analysis of diabetes and infectious disease management was not obtained. A cost analysis was also not performed, but recent reports have described improved costs for Apligraf versus TWC when treating CVI and DFU.^43,44 Chang et al. estimated a single Apligraf application costs $7,000–$10,000 less than a traditional split thickness skin graft (STSG) when considering the multiple hospital and operating room costs for STSG as compared to an outpatient Apligraf application.³⁸ It seems feasible that these cost reductions could also be available to this CLI patient population, especially when considering the high economic burden of treating CLI and amputations placed on our global healthcare system.^5,39

pligraf substantially decreased the time to WH in our CLI patients as compared to TWC (mean, 51 versus 83 days). There were no infections related to the Apligraf and we believe that early Apligraf application to the CLI wound (immediate, 48 hours) is of importance to WH by providing a physical and biological barrier against wound infection and desiccation, and immediately stimulating WH. The overall 6-month LS rate of 86.8% is consistent with the LACI trial results and several TBS reports, even in this truly end-stage CLI patient population with Rutherford Class 5 and 6 disease. Even though the study sample size was small, the Apligraf group also had a substantially improved 6-month LS rate as compared to TWC (92.3% versus 79.3%). Another important clinical advantage of Apligraf was improved pain control versus TWC even after revascularization. This benefit has been described before and the exact mechanism of pain control remains undefined and may reflect fewer dressing changes or earlier WH.^27,45 The once weekly dressing change protocol for the first 3 weeks has potential advantages, including simplicity, improved patient satisfaction, less pain, less healthcare resource utilization and time, at lower costs.

Conclusion

CLI is rapidly becoming a global epidemic and is responsible for > 220,000 amputations and > $20 billion in economic costs yearly in the U.S. and Europe. Optimization of multidisciplinary treatment will be necessary if we are to make a positive impact in treating CLI. It is time that all disciplines involved with the diagnosis and treatment of CVI, DFU and CLI patients open communications and lower their thresholds for novel treatments to optimize outcomes. In that spirit, and in the spirit of multidisciplinary education, the inaugural international multidisciplinary CLI Summit was held in Miami, Florida, October 26–27, 2005, with an attendance of approximately 400 individuals. The combination of ELA and Apligraf was found to substantially improve time to WH versus TWC in our CLI patient population. At 6 months, LS rates were also found to be improved in the ELA-Apligraf group versus the revascularization group, with TWC (92.3% versus 79.3%). A larger prospective multicenter randomized trial is warranted.

Acknowledgement The authors wish to thank Mrs. Kelly Tilbe, NCMA for her technical help with manuscript preparation.