Application of Chitosan and Chondroitin Sulphate Aerogels in a Patient With Diabetes With an Open Forefoot Transmetatarsal Amputation

Introduction. Diabetic foot ulcers may lead to nontraumatic amputations of the foot, leading to a decrease in patient quality of life. Transmetatarsal amputations (TMAs) represent an effective surgical procedure in cases of severe foot infection, but the tissue reconstruction is complicated and additional procedures should be considered. The present case report evaluates the wound closure of an open TMA in a patient with diabetes treated with a new aerogel composed of chitosan (ChS) and chondroitin sulphate (CS), without needing a skin graft. Case Report. A 72-year-old man with diabetes and a history of successive amputations was admitted to a hospital in Valdivia, Chile, due to a severe infection of toes 2 and 4 of the right foot. After the diagnosis of gangrene and osteomyelitis, the patient underwent a TMA of his right forefoot. The surgeon proposed the incorporation of ChS and CS aerogels to accelerate wound healing to avoid another surgical procedure. The TMA surgical wound area closed 50% after day 28 from starting treatment with aerogels. Complete closure was achieved at day 94 of treatment with aerogels, with good epithelial tissue and favorable cosmetic results and without residual limb deformities. The patient experienced minimal physical and psychological impairment from the procedure. Other surgical procedures were not necessary. Conclusions. Due to the results of this patient, use of ChS and CS aerogels could represent an alternative treatment for forefoot TMA wound closure and prevent further surgical procedures, such as skin grafting. Future works should consider a larger number of cases.

Diabetic foot ulcers (DFUs) are a complication of diabetes mellitus that may lead to nontraumatic amputations of the foot, with a negative impact on patient quality of life and economic burden.^1,2 Tissue reconstruction in patients with foot amputations is complicated. Good surgical handling of soft tissue should be considered, with the goal of maintaining a functional and plantigrade limb with good cosmetic and functional results. However, wound healing is frequently a major challenge. Hence, alternative procedures and new wound healing materials should be considered for this type of wound.^1-3 

Transmetatarsal amputations (TMAs) represent an effective surgical procedure in cases of severe forefoot infection and gangrene. Despite their low healing rate, if there is gangrene involving more than 2 toes, a TMA is considered by many surgeons to be the operation of choice.² It preserves foot function and is cosmetically favorable.^2,3 Patients with a healed TMA have good mobility and independent ambulation.⁴ 

All amputations require meticulous preoperative and postoperative management in order to prevent frequent wound healing failure and reduce the high economic cost due to prolonged hospitalization, rehabilitation, and home care.² Thus, efforts to guarantee the best treatment that ensures wound healing are warranted.⁵ Reconstructive techniques such as skin grafting could be used after forefoot TMA, permitting a fast covering, preventing infections, and providing psychological relief for these patients⁶; however, some patients can experience anxiety and mood changes after a skin graft.⁷ Sometimes skin grafting requires an extensive period of wound bed preparation, which in some cases can be up to 40 days.⁶ Donor site wounds also require care during the postoperative period, and some drawbacks include scarring and poor cosmetic outcome.⁸ 

The authors have previously reported the development of a new investigational material composed of chitosan (ChS) and chondroitin sulphate (CS) aerogels.⁹ Chitosan is a natural polymer widely used in the biomedical field, is biocompatible and biodegradable, and has antimicrobial effects.¹⁰ In combination with the glycosaminoglycan (GAG) of the extracellular matrix (ECM), such as CS, hyaluronic acid, and dermatan sulfate, it can form sponges and scaffold for diverse applications in tissue engineering, chronic wound healing, and drug delivery.¹¹ 

The aerogel used here is cell-free, developed in an aqueous medium, and uses a low concentration of reagents in their composition. This aerogel was designed as a skin wound healing agent with the goal to minimize the amount of matter applied to wounds.⁹ The lower density of the materials allows for better control of the individual polymer components of ChS and CS and presents better biological properties for several applications and minor metabolic stress in the damaged tissue.¹² In addition, the molecular assembling of this material based in electrostatic forces avoids the use of cross-linkers frequently related with toxic and allergic reactions.¹³ Diverse nanoformulations have shown potential therapeutic effects on wound healing and allow achieving a better control of the dosage.¹⁴ Aerogels synthesized under these criteria are biocompatible and provide specific properties for the induction of wound healing, do not affect the metabolic activity of cultured cells, and in experimental animal models, open wounds close significantly faster, unlike control wounds.¹⁵ The ability demonstrated by ChS and CS aerogels to induce wound closure also was observed by the authors in a preliminary study of 3 patients with diabetes who had undergone amputation of one of their toes.¹⁶

The present case reports the wound closure of an open TMA in a patient with diabetes who was treated with ChS and CS aerogels and did not need additional surgical procedures. 

The case of a 72-year-old man undergoing treatment for hypertension and type 2 diabetes mellitus and with a history of multiple hospitalizations for DFUs, progressive amputations on toes 1 and 2 of the left foot and 1 and 5 of the right foot, and femoropopliteal revascularization of the lower left limb is presented. Of note, 6 months before the present issue, the patient was admitted to the surgery department with an infected right plantar ulcer. He showed clinical signs of active infection of soft tissue with necrosis, gangrene, and osteomyelitis on toes 2 and 4 of the right foot. The patient had a palpable pedal pulse and an ankle-brachial index of 1.1 on the right leg. Surgical debridement and open TMA of the right forefoot were carried out, and the patient underwent antibiotic therapy of ceftriaxone 1 g daily and metronidazole 500 mg every 8 hours for 1 week. After finishing the antibiotic treatment, the patient was discharged in a wheelchair with prohibition to walk in order to relieve pressure and protect the wound area; the nurse clearly explained to the patient to not walk or stand on his feet. Subsequently, the patient remained under ambulatory wound care with standard dressing. The vascular surgeon proposed treatment with ChS and CS aerogels to accelerate healing and not subject the patient to another procedure (eg, skin graft) due to the psychological distress that he presented (ie, anxiety, stress, lowered mood, and low self-esteem). Written informed consent was obtained in accordance with the ethical principles of the Helsinki Declaration and approval of the Ethical Committee of Health Service in Valdivia, Chile. 

Wound care protocol
The wound bed was cleansed for 2 minutes with saline (0.9% w/ NaCl) and then gauze soaked with 0.1% betaine polyhexanide was applied for 10 minutes. Subsequently, sterile pieces of ChS and CS aerogels with a diameter of 2.5 cm (Figure 1) were placed directly over the wound bed. The wound then was covered using standard dressings, as hydrophilic foams to control the exudate or soft silicon to protect the wound, conventional gauze, and wrapped with a layer of Kling (Johnson & Johnson, New Brunswick, NJ). Once in the wound bed, the aerogels suffer hydration being rapidly absorbed. Dressing changes and ChS and CS aerogels applications were done twice weekly. The frequency of applications of aerogels were reduced to once weekly after wound closure reached 50%. The patient was monitored up until wound closure. The wound was photographed, and its margins were drawn weekly with an indelible marker over a sterile transparent film. The images were digitalized and uploaded to AutoCAD 2009 software (Autodesk, Mill Valley, CA) to determine their area. The total wound closure was recognized when full epithelialization was achieved with the absence of drainage.

At the time of initiating treatment with ChS and CS aerogels, the surgical wound area was 37.87 cm² (Figure 2) and presented an extensive raw surface with different exposed tissues. The outstanding improvement was evident as soon as treatment began, with 75% to 90% of granulation tissue and reepithelialization from the wound margins (Figure 3). After 28 days of aerogel treatment, the surgical wound area was 50% closed. Complete closure was achieved after 94 days of aerogel treatment (Figure 2), with good epithelial tissue and good cosmetic results, without residual limb deformities and without depressions, slits, or deformations. The skin did not present irregularities, hyperkeratosis, or stiffness. There were no complications in surgical wound healing during treatment with aerogels, such as infection, pain, or problems associated to surrounding skin. Furthermore, good care of the wound bed, relif due to offloading pressure from the foot, and the patient's cooperation allowed wound closure without complications. The epithelialization covered the wound bed from the borders with a smooth, good quality surface and without stump deformities (Figure 3). The patient’s mood improved greatly (ie, he was happy with the fast wound healing and ability to avoid undergoing another surgery). 

At 7 days following the end of treatment, the patient started wearing full-length therapeutic footwear with a total-contact insert and rigid rocker-bottom sole made by a physiotherapist. At the 36-month follow-up, the cicatrization of the stump persisted with excellent skin state (Figure 4), and the patient still had a functional plantigrade limb. 

Diabetic foot wounds, particularly those secondary to amputation, are typically complex, difficult to treat, and difficult to predict successful healing. A TMA is a surgical option performed in cases of severe forefoot infection, but only about 50% of patients heal^4,17 and not all achieve independent ambulation and good foot mobility.⁴ Wounds from TMAs that do not heal undergo subsequent complications such as postoperative infection, chronic stump ulceration, stump deformity, and additional proximal amputations.^18,19 Time to heal is often lengthy, with a mean of 7 to 8 months to up to 20 months, making the patients endure months of dressing changes and discomfort.²⁰ Two studies^20,21 reported only a 46% to 62% rate of success on healing after TMA procedures in patients with diabetes, with a mean healing time of 7 to 8 months, respectively. The reconstructive technique using skin grafting for larger wounds permits a fast covering to prevent infection but requires extensive grafting preparation periods, produces an additional wound and costs related to donor site intervention, and extends the hospitalization period.⁶ Yeh et al⁶ reported a wound healing rate of 86% in 2 months after the skin graft, with a mean total hospitalization time of 53 days. 

In the current case report, over a 3-month period using ChS and CS aerogels and standard dressing therapies, full wound closure was achieved. The surgical wound area of 37.87 cm² improved by 50% closure at 28 days until full closure and favorable cosmetic results. Despite the initial therapeutic indication of the graft in the present patient, the remarkable healing of the surgical wound allowed the vascular surgeon to revoke the graft indication. No hospitalization, anesthesia, or additional surgery was required, allowing a better adherence to the treatment and improvement in the emotional state of the patient. After 36 months of follow-up, stump deformity and wound recurrence were not present. 

Previously, Vidal et al¹⁶ reported the use of ChS and CS aerogels in smaller surgical wounds of 1.7 cm² to 6.4 cm² in 3 patients with DFUs who underwent a minor amputation of the toes and without indication of skin graft. Wound closure was achieved in 42 to 60 days.¹⁶ In the current case report, the surgical wound area was 6 times larger than noted by Vidal et al¹⁶ and complete healing was reached in 94 days without complications or another surgery. In their clinical practice, the present authors have used ChS and CS aerogels in DFUs and venous stasis wounds and observed an improvement of the vascular bed, mainly those wounds with necrotic tissue, and a shortening of wound closure times. 

Wound healing devices are often engineered as scaffolds of high structural stability, with interstices that allow aqueous flow and cell migration.¹¹ Such devices have been mainly developed to treat loss of solid tissues or extensive wounds. In contrast, the ChS and CS aerogel is hydrated and incorporated rapidly into the wound bed as shown by traces that remain after 48 hours of implantation. Further, the aerogel induces a speedy initial wound closure, which is followed over the subsequent days or weeks by a slower process, as can be observed in experimental animal models¹⁵ and human patients.¹⁶ This outcome may be due to the lower amount of material used and with a booster over a putative cellular reserve represented by steady state cells in treated tissues. Interestingly, animal models with 1 application of the aerogel have shown complete reepithelialization at day 14.¹⁵ Additionally, in the dermis, scar formation occurred with fibroblast, collagen, and neovascularization reduction, while control wounds showed epithelialization and scarring still in progress.¹⁵ However, the authors noted a diffuse inflammatory reaction in the dermis.¹⁵ In connection with these results, Kojima et al²² reported an increase of collagen in rat wounds treated with ChS, one of the components of the product used herein.

The aerogels could operate at the intercellular microenvironment over the ongoing bidirectional interplay among cells and their surrounding microenvironment.²³ Varied functions have been described for GAGs in living tissues, such as their capacity to operate on cell behavior, regulating molecules that are able to guide cells through chemoattraction, and bonding or enhancing the increase of environmental cytokine concentrations, growth factors, and other soluble active proteins in the ECM.²³ The fibroblast growth factor (FGF) family, a key player in the regulation of angiogenesis, is an example of a growth factor that must be bound to the ECM to exert its effects, interacting with heparin-like moieties in the ECM and the cellular plasma membrane in order to stimulate target cells throughout the phases of wound healing.^23,24 In effect, FGF-2 and keratinocyte growth factor/FGF-7 (ie, 2 members of the FGF family) promote proliferative processes and stimulate reepithelialization during wound healing by their preferential bond to iduronic acid motifs in ChS or dermatan sulfate.^24,25 In addition, Gómez et al²⁶ reported antibacterial action of this aerogel against Staphylococcus aureus, a recurrent pathogenic microorganism in chronic skin lesions; the potential antibacterial effect can be reinforced when the ChS and CS aerogel is loaded with erythromycin or elephant garlic extract.²⁶

Currently, sophisticated treatments commonly involve high financial costs and insufficient evidence of their cost-effectiveness.²⁷ This ChS and CS aerogel is a low-cost material, composed from biodegradable and biocompatible compounds that is easy to manufacture and store.⁹ The production cost of aerogels for this patient’s 3 months of treatment amounted to $30. Also, the aerogel does not contain proteins, their mechanical properties facilitate handling and application on open wounds, can be easily adapted to the wound contour due to the low toughness and hydration ability, and can be applied in the outpatient care setting. 

Finally, it is important to highlight the evident progress in the wound healing of the present patient since starting treatment with ChS and CS aerogels, having no complications, showing good cosmetic results of the stump, and having a functional plantigrade limb, which permitted a significant improvement in the patient’s quality of life. 

Despite obtaining satisfactory results, the present case study has 3 important limitations. First, outcomes of a single case could be suggestive but not indicative enough of the potential efficacy for ChS and CS aerogels in wound healing induction to prevent additional surgeries (eg, skin grafting). Hence, further research with an emphasis on larger case studies involving forefoot TMAs is needed. In a more general sense, curative efficacy of the product demand their study also in patients without diabetes who deal with TMAs. Second, to an adequacy of the evaluation of aerogels efficacy, the present study has no control group treated only with standard or advanced wound dressings. For this reason, cost efficacy studies are also required. A third limitation in this case was that the aerogels were used in a limited number of pieces; therefore, more research is necessary to ensure their effectiveness after mass production and the different origins of the raw materials. Other clinical variables, such as glycemic control and comorbidities, should be considered.

Diabetic foot ulcers and limb amputations often cause a great emotional impact on patients, especially if they underwent previous amputation, as seen in the current case. From the results of the present case, the use of ChS and CS aerogels could represent an alternative treatment for forefoot TMA wound closure in patients with diabetes and prevent additional surgical procedures, therefore improving patient quality of life; however, further research is needed to confirm this. This product is biocompatible, low cost, and easy to handle for topical applications in outpatient care. Future works should consider a larger number of cases of forefoot TMA treated with ChS and CS aerogels. 

Note: The authors thank Mrs. Claudia Lopez andKatherina Delgado for providing professional nursing assistance. 

Authors: Alejandra Vidal, MD¹; Annesi Giacaman, PhD²; Sandra L. Orellana, PhD³; Sandra Jofré, RN⁴; Ignacio Moreno-Villoslada, PhD³; Felipe Oyarzún-Ampuero, PhD⁵; and Miguel Concha, PhD¹

Affiliations: ¹Instituto de Anatomía, Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile; ²Centro Jeffrey Modell, para Diagnóstico e Investigación en Inmunodeficiencias Primarias, Centro de Excelencia en Medicina Traslacional, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile; ³Instituto de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile; ⁴Centro de Salud Familiar Rural de Niebla, Valdivia, Chile; and ⁵Departamento de Ciencias y Tecnologías Farmacéuticas, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile 

Correspondence: Alejandra Vidal, MD, Instituto de Anatomía, Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile; avidal@uach.cl 

Disclosure: Financial support of this work was provided by INNOVA Chile No. 07-CN13 IBM 252 and Regional Government of Los Ríos (FIC-R) Chile, No. 12-117.