Diabetic foot ulcer (DFU) is a chronic wound frequently delayed from severe infection. Wound dressing provides an essential barrier between the ulcer and the external environment. This review aimed to analyse the effectiveness of antibacterial collagen-based dressing for DFU treatment in a clinical setting. An electronic search in four databases, namely, Scopus, PubMed, Ovid MEDLINE(R), and ISI Web of Science, was performed to obtain relevant articles published within the last ten years. The published studies were included if they reported evidence of (1) collagen-based antibacterial dressing or (2) wound healing for diabetic ulcers, and (3) were written in English. Both randomised and non-randomised clinical trials were included. The search for relevant clinical studies (n) identified eight related references discussing the effectiveness of collagen-based antibacterial wound dressings for DFU comprising collagen impregnated with polyhexamethylene biguanide (n = 2), gentamicin (n = 3), combined-cellulose and silver (n = 1), gentian violet/methylene blue mixed (n = 1), and silver (n = 1). The clinical data were limited by small sample sizes and multiple aetiologies of chronic wounds. The evidence was not robust enough for a conclusive statement, although most of the studies reported positive outcomes for the use of collagen dressings loaded with antibacterial properties for DFU wound healing. This study emphasises the importance of having standardised clinical trials, larger sample sizes, and accurate reporting for reliable statistical evidence confirming DFU treatment efficiency.
Gelatin possesses biological properties that resemble native skin and can potentially be fabricated as a skin substitute for full-thickness wound treatment. The native property of gelatin, whereby it is easily melted and degraded at body temperature, could prevent its biofunctionality for various applications. This study aimed to fabricate and characterise buffalo gelatin (Infanca halal certified) crosslinked with chemical type crosslinker (genipin and genipin fortified with EDC) and physicaly crosslink using the dihydrothermal (DHT) method. A porous gelatin sponge (GS) was fabricated by a freeze-drying process followed by a complete crosslinking via chemical-natural and synthetic-or physical intervention using genipin (GNP), 1-ethyl-3-(3-dimethylaminopropyl) (EDC) and dihydrothermal (DHT) methods, respectively. The physicochemical, biomechanical, cellular biocompatibility and cell-biomaterial interaction of GS towards human epidermal keratinocytes (HEK) and dermal fibroblasts (HDF) were evaluated. Results showed that GS had a uniform porous structure with pore size ranging between 60 and 200 µm with high porosity (>78.6 ± 4.1%), high wettability (<72.2 ± 7.0°), high tensile strain (>13.65 ± 1.10%) and 14 h of degradation rate. An increase in the concentration and double-crosslinking approach demonstrated an increment in the crosslinking degree, enzymatic hydrolysis resistance, thermal stability, porosity, wettability and mechanical strength. The GS can be tuned differently from the control by approaching the GS via a different crosslinking strategy. However, a decreasing trend was observed in the pore size, water retention and water absorption ability. Crosslinking with DHT resulted in large pore sizes (85-300 µm) and low water retention (236.9 ± 18.7 g/m2·day) and a comparable swelling ratio with the control (89.6 ± 7.1%). Moreover no changes in the chemical content and amorphous phase identification were observed. The HEK and HDF revealed slight toxicity with double crosslinking. HEK and HDF attachment and proliferation remain similar to each crosslinking approach. Immunogenicity was observed to be higher in the double-crosslinking compared to the single-crosslinking intervention. The fabricated GS demonstrated a dynamic potential to be tailored according to wound types by manipulating the crosslinking intervention.
The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.
The use of gelatin microspheres (GMs) as a cell carrier has been extensively researched. One of its limitations is that it dissolves rapidly in aqueous settings, precluding its use for long-term cell propagation. This circumstance necessitates the use of crosslinking agents to circumvent the constraint. Thus, this study examines two different methods of crosslinking and their effect on the microsphere's physicochemical and cartilage tissue regeneration capacity. Crosslinking was accomplished by physical (dehydrothermal [DHT]) and natural (genipin) crosslinking of the three-dimensional (3D) GM. We begin by comparing the microstructures of the scaffolds and their long-term resistance to degradation under physiological conditions (in an isotonic solution, at 37°C, pH = 7.4). Infrared spectroscopy indicated that the gelatin structure was preserved after the crosslinking treatments. The crosslinked GM demonstrated good cell adhesion, viability, proliferation, and widespread 3D scaffold colonization when seeded with human bone marrow mesenchymal stem cells. In addition, the crosslinked microspheres enhanced chondrogenesis, as demonstrated by the data. It was discovered that crosslinked GM increased the expression of cartilage-related genes and the biosynthesis of a glycosaminoglycan-positive matrix as compared with non-crosslinked GM. In comparison, DHT-crosslinked results were significantly enhanced. To summarize, DHT treatment was found to be a superior approach for crosslinking the GM to promote better cartilage tissue regeneration.
The irregular shape and depth of wounds could be the major hurdles in wound healing for the common three-dimensional foam, sheet, or film treatment design. The injectable hydrogel is a splendid alternate technique to enhance healing efficiency post-implantation via injectable or 3D-bioprinting technologies. The authentic combination of natural and synthetic polymers could potentially enhance the injectability and biocompatibility properties. Thus, the purpose of this study was to characterise a hybrid gelatin−PVA hydrogel crosslinked with genipin (GNP; natural crosslinker). In brief, gelatin (GE) and PVA were prepared in various concentrations (w/v): GE, GPVA3 (3% PVA), and GPVA5 (5% PVA), followed by a 0.1% (w/v) genipin (GNP) crosslink, to achieve polymerisation in three minutes. The physicochemical and biocompatibility properties were further evaluated. GPVA3_GNP and GPVA5_GNP with GNP demonstrated excellent physicochemical properties compared to GE_GNP and non-crosslinked hydrogels. GPVA5_GNP significantly displayed the optimum swelling ratio (621.1 ± 93.18%) and excellent hydrophilicity (38.51 ± 2.58°). In addition, GPVA5_GNP showed an optimum biodegradation rate (0.02 ± 0.005 mg/h) and the highest mechanical strength with the highest compression modulus (2.14 ± 0.06 MPa). In addition, the surface and cross-sectional view for scanning electron microscopy (SEM) displayed that all of the GPVA hydrogels have optimum average pore sizes (100−199 μm) with interconnected pores. There were no substantial changes in chemical analysis, including FTIR, XRD, and EDX, after PVA and GNP intervention. Furthermore, GPVA hydrogels influenced the cell biocompatibility, which successfully indicated >85% of cell viability. In conclusion, gelatin−PVA hydrogels crosslinked with GNP were proven to have excellent physicochemical, mechanical, and biocompatibility properties, as required for potential bioinks for chronic wound healing.
Gelatin usage in scaffold fabrication is limited due to its lack of enzymatic and thermal resistance, as well as its mechanical weakness. Hence, gelatin requires crosslinking and reinforcement with other materials. This study aimed to fabricate and characterise composite scaffolds composed of gelatin, elastin, and cellulose nanocrystals (CNC) and crosslinked with genipin. The scaffolds were fabricated using the freeze-drying method. The composite scaffolds were composed of different concentrations of CNC, whereas scaffolds made of pure gelatin and a gelatin-elastin mixture served as controls. The physicochemical and mechanical properties of the scaffolds, and their cellular biocompatibility with human dermal fibroblasts (HDF), were evaluated. The composite scaffolds demonstrated higher porosity and swelling capacity and improved enzymatic resistance compared to the controls. Although the group with 0.5% (w/v) CNC recorded the highest pore size homogeneity, the diameters of most of the pores in the composite scaffolds ranged from 100 to 200 μm, which is sufficient for cell migration. Tensile strength analysis revealed that increasing the CNC concentration reduced the scaffolds' stiffness. Chemical analyses revealed that despite chemical and structural alterations, both elastin and CNC were integrated into the gelatin scaffold. HDF cultured on the scaffolds expressed collagen type I and α-SMA proteins, indicating the scaffolds' biocompatibility with HDF. Overall, the addition of elastin and CNC improved the properties of gelatin-based scaffolds. The composite scaffolds are promising candidates for an acellular skin substitute.
Chronic wounds are challenging to heal and increase global mortality. The effectiveness of skin graft is limited by rejection, fibrosis, and inadequate donor site. Multifunctionalised-hydrogel skin substitutes promoted higher wound healing by maintaining the moisture microenvironment and permit gas exchange/nourishment in prolong cell viability/activity. The purpose of this study was to evaluate a skin substitute using two strategies; via injectable and 3D bioprinting technique. New hydrogel formulations that composed of gelatin (GE) and polyvinyl-alcohol (PVA) were constructed using a pre-mix crosslinking approach with genipin (GNP) to generate the biodegradable and biocompatible skin substitute with reduced secondary traumatic wound. GPVA5_GNP (6% GE: 5% PVA crosslinked with GNP) was the most stable hydrogel for wound healing application with the longest enzymatic degradation and stable hydrogel for absorption of excess wound exudates. Primary human dermal fibroblasts (HDFs) migrated extensively through 3D bioprinted hydrogels with larger average pore sizes and interconnected pores than injectable hydrogels. Moreover, 3D bioprinted GPVA hydrogels were biocompatible with HDFs and demonstrated > 90% cell viability. HDFs maintained their phenotype and positively expressed collagen type-I, vinculin, short and dense F-actin, alpha-smooth muscle actin, and Ki67. Additionally, the presence of GNP demonstrated antioxidant capacity and high-ability of angiogenesis. The utilisation of the 3D bioprinting (layer-by-layer) approach did not compromise the HDFs' growth capacity and biocompatibility with selected bioinks. In conclusion, it allows the cell encapsulation sustainability in a hydrogel matrix for a longer period, in promoting tissue regeneration and accelerating healing capacity, especially for difficult or chronic wound.
The irregular deep chronic wound is a grand challenge to be healed due to multiple factors including slow angiogenesis that causing regenerated tissue failure. The narrow gap of deep wounds could hinder and slow down normal wound healing. Thus, the current study aimed to develop a polymerised genipin-crosslinked gelatin (gelipin) hydrogel (GNP_GH) as a potential biodegradable filler for the abovementioned limitations. Briefly, GNP_GH bioscaffolds have been developed successfully within three-minute polymerisation at room temperature (22-24 °C). The physicochemical and biocompatibility of GNP_GH bioscaffolds were respectively evaluated. Amongst GNP_GH groups, the 0.1%GNP_GH10% displayed the highest injectability (97.3 ± 0.6%). Meanwhile, the 0.5%GNP_GH15% degraded within more than two weeks with optimum swelling capacity (108.83 ± 15.7%) and higher mechanical strength (22.6 ± 3.9 kPa) than non-crosslinked gelatin hydrogel 15% (NC_GH15%). Furthermore, 0.1%GNP_GH15% offered higher porosity (>80%) and lower wettability (48.7 ± 0.3) than NC_GH15%. Surface and cross-section SEM photographs displayed an interconnected porous structure for all GNP_GH groups. The EDX spectra and maps represented no major changes after GNP modification. Moreover, no toxicity effect of GNP_GH against dermal fibroblasts was shown during the biocompatibility test. In conclusion, the abovementioned findings indicated that gelipin has excellent physicochemical properties and acceptable biocompatibility as an acellular rapid treatment for future use in irregular deep cutaneous wounds.
Nanotechnology has become an emerging technology in the medical field and is widely applicable for various clinical applications. The potential use of nanoparticles as antimicrobial agents is greatly explored and taken into consideration as alternative methods to overcome the challenges faced by healthcare workers and patients in preventing infections caused by pathogenic microorganisms. Among microorganisms, bacterial infections remain a major hurdle and are responsible for high morbidity and mortality globally, especially involving those with medical conditions and elderly populations. Over time, these groups are more vulnerable to developing resistance to antibiotics, as bacterial biofilms are difficult to destroy or eliminate via antibiotics; thus, treatment becomes unsuccessful or ineffective. Mostly, bacterial biofilms and other microbes can be found on medical devices and wounds where they disperse their contents which cause infections. To inhibit biofilm formations and overcome antibiotic resistance, antimicrobial-loaded nanoparticles alone or combined with other substances could enhance the bactericidal activity of nanomaterials. This includes killing the pathogens effectively without harming other cells or causing any adverse effects to living cells. This review summarises the mechanisms of actions employed by the different types of nanoparticles which counteract infectious agents in reducing biofilm formation and improve antibiotic therapy for clinical usage.
Recent advancement in cartilage tissue engineering has explored the potential of 3D culture to mimic the in vivo environment of human cartilaginous tissue. Three-dimensional culture using microspheres was described to play a role in driving the differentiation of mesenchymal stem cells to chondrocyte lineage. However, factors such as mechanical agitation on cell chondrogenesis during culture on the microspheres has yet to be elucidated. In this study, we compared the 2D and 3D culture of bone-marrow-derived mesenchymal stem cells (BMSCs) on gelatin microspheres (GMs) in terms of MSC stemness properties, immune-phenotype, multilineage differentiation properties, and proliferation rate. Then, to study the effect of mechanical agitation on chondrogenic differentiation in 3D culture, we cultured BMSCs on GM (BMSCs-GM) in either static or dynamic bioreactor system with two different mediums, i.e., F12: DMEM (1:1) + 10% FBS (FD) and chondrogenic induction medium (CIM). Our results show that BMSCs attached to the GM surface and remained viable in 3D culture. BMSCs-GM proliferated faster and displayed higher stemness properties than BMSCs on a tissue culture plate (BMSCs-TCP). GMs also enhanced the efficiency of in-vitro chondrogenesis of BMSCs, especially in a dynamic culture with higher cell proliferation, RNA expression, and protein expression compared to that in a static culture. To conclude, our results indicate that the 3D culture of BMSCs on gelatin microsphere was superior to 2D culture on a standard tissue culture plate. Furthermore, culturing BMSCs on GM in dynamic culture conditions enhanced their chondrogenic differentiation.
The eminent aim for advance wound management is to provide a great impact on the quality of life. Therefore, an excellent strategy for an ideal wound dressing is being developed that eliminates certain drawbacks while promoting tissue regeneration for the prevention of bacterial invasion. The aim of this study is to develop a bilayer hybrid biomatrix of natural origin for wound dressing. The bilayer hybrid bioscaffold was fabricated by the combination of ovine tendon collagen type I and palm tree-based nanocellulose. The fabricated biomatrix was then post-cross-linked with 0.1% (w/v) genipin (GNP). The physical characteristics were evaluated based on the microstructure, pore size, porosity, and water uptake capacity followed by degradation behaviour and mechanical strength. Chemical analysis was performed using energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectrophotometry (FTIR), and X-ray diffraction (XRD). The results demonstrated a uniform interconnected porous structure with optimal pore size ranging between 90 and 140 μm, acceptable porosity (>70%), and highwater uptake capacity (>1500%). The biodegradation rate of the fabricated biomatrix was extended to 22 days. Further analysis with EDX identified the main elements of the bioscaffold, which contains carbon (C) 50.28%, nitrogen (N) 18.78%, and oxygen (O) 30.94% based on the atomic percentage. FTIR reported the functional groups of collagen type I (amide A: 3302 cm-1, amide B: 2926 cm-1, amide I: 1631 cm-1, amide II: 1547 cm-1, and amide III: 1237 cm-1) and nanocellulose (pyranose ring), thus confirming the presence of collagen and nanocellulose in the bilayer hybrid scaffold. The XRD demonstrated a smooth wavy wavelength that is consistent with the amorphous material and less crystallinity. The combination of nanocellulose with collagen demonstrated a positive effect with an increase of Young's modulus. In conclusion, the fabricated bilayer hybrid bioscaffold demonstrated optimum physicochemical and mechanical properties that are suitable for skin wound dressing.
The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis.