Wound healing and Metabolite Profiling in Collagen- Chitosan Biomaterial-treated Chronic Wounds of Hansen’s Disease Patients

Aims: Impaired wound healing causes chronic ulcers in Hansen’s disease (HD) patients which are an unrecognized clinical manifestation and requires utmost care and attention for wound management. Collagen and chitosan biopolymers when synergistically combined produce a biologically active biomaterial for wound dressings. Hence, the aim was to prepare a collagen/chitosan (COL/CS) composite and characterize for wound healing potential in HD patients. Place and Duration of Study: CSIR-Central Leather Research Institute, Sardar Patel Road, Adyar, Chennai 600021, Southern Railway Headquarters Hospital, Constable Road, Ayanavaram, Chennai 600023, and Gremaltes Hospital, India between June 2013 and July 2020. Methodology: The HD wounds were measured by Planimetry in square cm and were also Original Research Article Babu et al.; JPRI, 33(53A): 296-303, 2021; Article no.JPRI.78024 297 assessed for morphological structure of epidermis and collagen fiber arrangement by High ResolutionScanning electron microscopy (HR-SEM). Proton Nuclear magnetic resonance spectroscopy ( 1 H-NMR) for metabolite identification was studied in blood plasma samples of unwounded, untreated and treated HD patients Results: Size D (wound size on day of discharge) of the wounds were appreciably lower than Size 0 (wound size before biomaterial treatment) demonstrating efficient wound healing by the biomaterial. The morphological structure of the HD wounds showed healthy epidermal layer and thick fibers of collagen matrix in the treated wounds when compared to the controls. Key metabolites of metabolic pathways such as TCA cycle, creatine cycle and protein metabolism were identified by 1 H-NMR spectroscopy Conclusion: The COL/CS wound dressing is a promising biomaterial for management of chronic wounds in Hansen’s disease.

assessed for morphological structure of epidermis and collagen fiber arrangement by High Resolution-Scanning electron microscopy (HR-SEM). Proton Nuclear magnetic resonance spectroscopy ( 1 H-NMR) for metabolite identification was studied in blood plasma samples of unwounded, untreated and treated HD patients Results: Size D (wound size on day of discharge) of the wounds were appreciably lower than Size 0 (wound size before biomaterial treatment) demonstrating efficient wound healing by the biomaterial. The morphological structure of the HD wounds showed healthy epidermal layer and thick fibers of collagen matrix in the treated wounds when compared to the controls. Key metabolites of metabolic pathways such as TCA cycle, creatine cycle and protein metabolism were identified by 1

INTRODUCTION
Skin injury causes wounds which destroy the skin tissue organization and may lead to chronic conditions imposing economic burden and social concerns. Normal wound healing must occur sequentially in a specific manner, time and duration. Acute and chronic wounds cause microbial infection, loss of body fluids, electrolytes and nutrients [1]. In delayed acute wounds and chronic wounds, healing process is impaired and uncoordinated resulting in pathologic inflammation [2]. Wound management or treatment of wounds is required to restore the normal skin structure. Wound healing materials or wound dressings should ideally be antiinfective, hemostatic and exhibit histocompatibility, reduce wound healing time, side effects of drugs and improve bioavailability [3]. Further, the wound dressing should be flexible, biodegradable, keep wounds moist and adsorb exudates [4]. Traditional wound dressings such as cotton bandage or gauze absorb most of the moisture leaving the wound dry and decreasing the healing rate [5]. Natural biopolymers from animal origin such as collagen and chitosan are excellent biomaterials suited for wound dressings. These biopolymers are biocompatible, biodegradable, bioresorbable and promote cell adhesion and growth and tissue regeneration [6]. Collagen, in synergistic combination with chitosan [6,7] produces a biologically active material for wound dressings [8] which can promote cell proliferation [9] and angiogenesis [10] at the wound site.
Hansen's disease (HD) or leprosy as known earlier, is a debilitating illness and a public health problem. The wounds are hard-to-heal chronic plantar ulcers which require appropriate wound management. Wound dressings should be affordable and capable of reducing the healing time because long-term wound management could seriously affect the HD patients. Chronic ulcers in HD patients are an unrecognized clinical manifestation and need more attention on the treatment of HD ulcers [11,12]. Comorbid conditions are risk factors of delayed healing of plantar ulcers in HD patients [13]. Thus, there is a need to fabricate or develop suitable wound dressings for the treatment of chronic wounds in HD patients.
Considering these aspects, we have prepared a collagen/chitosan powder (COL/CS) from collagen extracted from bovine rumen and commercially available chitosan [14] and have studied its wound healing efficacy in chronic wounds of HD patients by wound size measurements and have examined the morphology of wound tissue by Scanning electron microscopy (SEM). The metabolite profiling of the patients' blood plasma were analysed by proton nuclear magnetic resonance ( 1 H-NMR) spectroscopy.

Patients
Human in-patients at Gremaltes Hospital, Chennai, India, were treated according to standard clinical guidelines.

Clinical Study
Patients with Hansen disease (HD) (n=65) were identified and included in the study. Their personal data, clinical data and past and present history were recorded. Deep (sinus) wounds were treated with collagen cream as these wounds cannot be penetrated by COL/CS powder, while, plantar and chronic wounds (Static) were treated with the COL/CS powder. Wound cleaning and debridement was done prior to the application of biomaterial. Wound dressings were done at intervals of four days. In this study, chronic wounds including amputations were also used as patients of treatment. Post treatment, wounds were periodically monitored and the images of the wound contour size were recorded. In a representative control, a patient was treated with T-Bact ointment. The ointment was applied on the wound and compared with the experimental wound. All the in-patients of the study were accommodated in clean and aseptic wards and were restricted from administering antibiotics of any type, in order to observe the effect of the biomaterial.

Planimetry
Wound size measurements of HD patients was done by planimetry method according to Babu et al. [14] and the size of the wound was calculated in square cm.

Morphology Examination by Scanning Electron Microscopy
Samples were cut out from the wound site at the wound edges of non HD (control) (n=4) and HD (test) (n=4) patients. The sample was then washed with a phosphate-buffered saline solution and freeze-dried by using lyophilizer (Lyovapor™ L-300, BUCHI, Switzerland). A small section of samples was attached to the stub, and the specimens were coated with a thin layer of gold ions in an Edwards vacuum coater with an offset rotating sample holder (for uniform coating of the irregular surface). The conducting path from the coating to the stub was done by the use of a conducting sticker. Samples were examined, and photomicrographs were recorded in the scanning electron microscope (HR-SEM, FEI; Quanta FEG 200, USA).

Metabolite profiling by 1 H-NMR Spectroscopy
In order to identify and quantify many metabolites in biological samples, NMR spectroscopy is a versatile technique to identify organic compounds based on determinations of molecular structures. Hence, the blood plasma which is composed of a number of known and unknown metabolites was used in this study. The unwounded patient sample was used as a representative blank (n=2). Patients with untreated wounds were studied as controls (n=2) and COL/CS-treated HD patients were investigated as the test samples (n=2). About 350 μL of plasma was mixed with 150 μL of deuterium oxide (D 2 O) and transferred into a 5 mm OD NMR sample tube. The 1 H-NMR acquisition was carried out on a 400 MHz highresolution BRUKER-Ascend TM-400 NB-NMR spectrometer with the following test parameters: 5 mm PABBO-BB probe and zg30 pulse program with 128 scans. The FID data was processed using BRUKER Topspin (v.3.2) software.

Statistical Analysis
Data were processed for Mean ± SD of 3 wound measurements of the sample and plotted by MS-Excel software v.2013.

Wound Healing Studies of COL/CS
Wound size measurements were done before (Size 0) and on the day of discharge (Size D) after application of COL/CS wound dressings. The data are represented in Fig.2 which shows the marked decrease on Size D values compared to Size 0 values of 65 patients. Planimetry studies clearly indicate the efficacy of COL/CS as a wound healing material. This dressing material composed of collagen and chitosan can be considered beneficial for wound management as collagen supports cell growth and tissue growth while, chitosan has red blood cells binding and antibacterial properties [15]. Tamer et al [16] have reported the depolymerisation of chitosan to release N-Acetyl glucosamine during the wound healing process. The N-Acetyl glucosamine has been shown to promote fibroblast proliferation, increase collagen matrix deposition and stimulate hyaluronic acid synthesis on the wound [17].
The surface morphology of wound tissue by HR-SEM is depicted in Fig.3 showing the epidermal layer (A, B) and the collagen fiber arrangement (C, D). The epidermis structure is distinguished in the fibrous tissue through the thickness of the dermis. The epidermis of the skin tissue has well developed and firm structure in COL/CS biomaterial-treated wound, but it is comparatively very less in control. Nearly underlying the epidermis and following its undulating form, is the layer of fine collagen fibers. There are thicker collagen fibers found in the test sample than the control sample. The porous structure of the wound dressing imparts roughness which improves cell affinity and increases the contact area of the dressing with the wound further accelerating hemostasis [18]. The microstructure of the dressing plays an important role in cellular attachment, migration and proliferation [19]. Our study therefore suggests the improved wound healing by COL/CS wound dressing in chronic wounds of HD patients.

Metabolite Profiling by NMR Spectroscopy
NMR spectroscopy is a highly versatile technique which shows specificity of spectra with respect to a particular metabolite and hence determines the structure of unknown compounds from biological samples [20]. By this strategy, the blood plasma samples of HD patients were processed and analysed. The unwounded patient sample was used as a representative blank (Fig.4a) and the reference peaks of metabolites were evaluated with untreated wounds as control (Fig.4b) and COL/CS-treated HD patients as the test sample (Fig.4c). Assignments of chemical shifts of unknown metabolites in the blood plasma to those reported in literature provide valuable information for the identification of the metabolites [21]. The identification of a new metabolite without standard spectral data are often a difficult task in the study of metabolome [22]. Therefore we have used an unwounded sample for a reference spectrum. The high presence of betaine in the COL/CS-treated sample suggests that this compound was overproduced to maintain normal function in the skin. The presence of betaine helps to maintain the normal physiological functions. The peaks of each spectrum correspond to the individual metabolite present in the circulating blood during healing. Therefore, these metabolites are considered biological markers in the healing of wounds. Many metabolites such as amino acids (alanine, glutamine, glycine and/or leucine/isoleucine), lipids and other energy metabolizing molecules (citrate, lactate, αketoglutarate, betaine, creatine, and creatinine) were identified in the plasma of wounded sample. Several metabolites of the tricarboxylic acid cycle, creatine cycle, and protein metabolism were identified in the NMR spectrum of plasma samples. The

CONCLUSION
Wound healing of chronic ulcers in Hansen's disease is a major condition to be addressed due to non-healing or long-term wound management causing alarming public health concern. We have prepared a suitable wound dressing composed of collagen/chitosan as a powder and have studied the wound healing efficacy by Planimetry and HR-SEM analyses and have further characterized the metabolic profiling of the HD patients' blood plasma by proton NMR spectroscopy. We have obtained good healing potential of the wound dressing by a significant decrease in wound size on the day of discharge and the morphology of the wound showed healthy epidermal layer and restructuring of the collagen matrix in the treated wounds. By NMR metabolite identification, several key intermediary metabolites were found to be in association with different metabolic pathways in the plasma samples of untreated and treated HD patients. These results were obtained from previous literature based on the chemical shifts of the unknown metabolites. Thereby, an effective wound dressing has been prepared from collagen-chitosan composite for treatment of chronic ulcers of HD patients.

CONSENT
Informed consents from the patients with Hansen disease were obtained prior to clinical investigations and observations. Appropriate controls were represented from non-Hansen disease patients (NHD).

ETHICAL APPROVAL
Ethical clearance and approval was obtained from Human Ethics Committee, Southern Railway Head Quarter Hospital, Perambur, Chennai, India. The proposal for conducting human clinical study was scrutinized and approved with the Approval No. SRHQH/EC 08112014. The study was performed according to the Declaration of Helsinki.