Faster, Better Healing: Galactomannans from Ceratonia siliqua as a Novel Solution for Wounded Skin

Wound healing is a universal issue of fundamental importance in the field of medical care. Globally, about 300 million patients are in fact afflicted with chronic skin lesions and 100 million patients have traumatic cutaneous wounds¹. One study has reported that among the 100 million new scars formed every year, about 10% become pathological². Thus, a deterioration of the healing process is a major concern for public health as this toll continues to grow. Consequently, promoting healing of damaged skin, restoring the functions of tissues and minimizing the esthetic impact of scars are a major challenge to clinical care^3–5.

From a biological standpoint, the healing process of the skin depends on three phases⁶. The inflammatory phase is the onset of the healing process and lasts about three days, with hemostasis and mobilization of immune defenses at the site of the wound. Neutrophils and macrophages infiltrate the wound and eliminate bacteria and debris by phagocytosis. Macrophages also participate in amplifying the inflammatory response required to commence the proliferation phase^5,7. 

Then, there is the proliferation phase that lasts about two weeks. This phase promotes repair of the vascular network and the development of new connective tissue and involves several biological processes. The proliferation phase requires the substantial activation of keratinocytes, fibroblasts, macrophages and endothelial cells to carry out re-epithelialization, deposit of the matrix and angiogenesis^3,8. During this phase, a reduction of inflammation also occurs to initiate the final phase of the healing process⁹. 

Finally, the remodeling phase is when granulation tissue matures into scar tissue. It can last several months to several years after the injury. In this phase, the dermal matrix is remodeled to promote the return to the initial state of the tissue’s architecture and resistance. Gradually, more substance is added to the extracellular matrix, a denser collagen structure appears, cellularity decreases, and the vascular network becomes organized^3,10,11.

Scientific progress in the field of healing has shown that the proliferation phase, involving granulation tissue formation, angiogenesis, re-epithelialization and inflammation resolution, is critical for tissue healing. Existing in vitro models often fail to replicate the complex interactions between vascular, immune, and cutaneous cells during the wound healing process, due to the challenges associated with co-cultivating these cell types. This research focuses on the development of an in vitro 3D skin model enabling to study skin healing dynamics and to substantiate the mechanism of action of a natural active ingredient. 

Experimental Design 

Development of a 3D Skin Model 

Normal human fibroblasts were seeded at 37°C for 28 days until dermal sheets formed. Normal human keratinocytes were then seeded on a portion of the dermal sheets and cultivated at 37°C for four days. Two dermal sheets and one dermis/epidermis sheet were layered. Endothelial and immune cells from the dermis of a donor were placed between the sheets. After ten days of culture at 37°C (D0), a vascularized immunocompetent 3D full thickness skin model was obtained (VI SILABSKIN FT). To mimic skin healing characteristics, it was later wounded with a biopsy punch. It was then systemically treated with a natural active ingredient (Galactomannans (GM) derived from Ceratonia siliqua) at 0.05% for seven days. 

Study of the Granulation Tissue Formation 

The secretion of growth factor TGF-β was analyzed on D3 post-injury by ELISA assay and the syntheses of the markers α-SMA, fibronectin, tenascin-C and collagen III were determined on D7 post-injury by immunohistofluorescence.

Study of the Vascular Network

The secretion of growth factor VEGF was analyzed on D3 post-injury with an ELISA assay. The density of the vascular network was determined on D7 post-injury by an immunohistofluorescence (marker CD31 specific to endothelial cells) and visualized by Light Sheet microscopy. 

Study of Inflammation

The inflammatory secretome was analyzed on D3 post-injury by ELISA assay and the population of M2 macrophages was studied on D7 post-injury using double labeling by immunohistofluorescence (CD206/CD68 labeling specific to M2 macrophages)¹² and visualized by Light Sheet microscopy. 

Study of Re-epithelialization 

The secretion of growth factors KGF and FGF was analyzed on D3 post-injury with an ELISA assay. The syntheses of markers of the basal membrane, of cohesion and of epidermal differentiation were determined on D7 post-injury by immunohistofluorescence. 

Results 

Formation of Granulation Tissue

Fibroblasts are responsible for the formation of granulation tissue. Due to their migration capacities, they enter the wound zone and proliferate there, primarily under the influence of growth factor TGF-β (Transforming growth factor-beta). In the presence of GM derived from Ceratonia siliqua, the secretion of TGF-β is increased by 18% (P < 0.001). A portion of the fibroblasts recruited specializes and differentiates into myofibroblasts characterized by increased expression of α-SMA, a protein conferring the acquisition of strong contractile properties. GM derived from Ceratonia siliqua increases the synthesis of α-SMA by 62% (P < 0.05). Figure 1 reveals that this active ingredient also increases tenascin-C, fibronectin and collagen III, three biological markers of the granulation tissue formation, by 19% (P < 0.01), 29% (P < 0.01) and 71% (P < 0.001) respectively.   

Figure 1 - Effect of GM derived from Ceratonia siliqua on the granulation tissue formation observed on wounded VI SILABSKIN FT. Courtesy of Silab

Action on the Vascularization

The cutaneous healing process requires vascularization of newly formed tissue. The number of blood vessels in the dermis increases as granulation tissue continues to form. GM derived from Ceratonia siliqua increases the secretion of VEGF by 19% (P < 0.001). Figure 2 reveals that this active ingredient also increases the density of the vascular network by 20% (P < 0.001). 

Figure 2 - Effect of GM derived from Ceratonia siliqua on the vascularization observed on wounded VI SILABSKIN FT. Courtesy of Silab

Reduction of Inflammation

The healing process is affected by a wide variety of pro-inflammatory molecules required to initiate the granulation tissue formation, angiogenesis and re-epithelialization. On the contrary, an excess of inflammatory factors may cause a delay in healing or even the formation of pathological scars. Among the cellular players involved in the control of inflammation, macrophages are transformed from a M1 pro-inflammatory phenotype in the inflammatory phase into a M2 anti-inflammatory pro-regenerating phenotype in the proliferation phase. This transition contributes to restoring functional cutaneous tissue and is indispensable for optimal healing ^9,13.

GM derived from Ceratonia siliqua decreases the secretion of the pro-inflammatory markers IL-8, IL-6, IL-1α and TNF-α by 37% (P < 0.001), 27% (P < 0.001), 36% (P < 0.05) and 24% (P < 0.001) respectively. Moreover, this active ingredient increases the presence of M2 macrophages by 37% (P < 0.05).

Figure 3 - Effect of GM derived from Ceratonia siliqua on the presence of pro-inflammatory M2 macrophages observed on wounded VI SILABSKIN FT. Courtesy of Silab

Re-epithelialization

Re-epithelialization is an essential biological mechanism to heal skin wounds by enabling regeneration of the epidermis. Keratinocytes play a predominant role in this process. They migrate, proliferate and differentiate to recreate a new functional epidermis solidly anchored in the newly formed basal membrane. The cellular and molecular processes participating in initiating, maintaining and completing re-epithelialization depend on growth factors such as KGF and FGF^7,14,15. 

According to the results, GM derived from Ceratonia siliqua increases the secretion of FGF and KGF by 55% (P < 0.01) and 15% (P < 0.01) respectively. Figure 3 shows that this active ingredient also improves epidermal cohesion and differentiation as well as the basal membrane organization. 

Figure 4 - Effect of GM derived from Ceratonia siliqua on re-epithelialization observed on wounded VI SILABSKIN FT. Courtesy of Silab

Discussion

The mechanism of action of a natural active ingredient (GM derived from Ceratonia siliqua) was demonstrated on an original in vitro 3D skin model that integrates the characteristics of the skin healing proliferation phase. Myofibroblasts guarantee the maturation of granulation tissue by synthesizing a new extracellular matrix primarily composed of collagen III and proteins required for repair of the dermis, such as fibronectin and tenascin-C¹⁶. Myofibroblasts bind to fibronectin and collagen fibrils, causing contraction of the wound. This indispensable phenomenon for the healing process reduces the surface of the wound to repair and improves the mechanical resistance of the surrounding tissues^7,8,10. 

Beyond their role as dermal repair factors, fibronectin and tenascin-C can regulate other biological mechanisms required in the healing process. They in fact promote angiogenesis and re-epithelialization of the wound. Tenascin-C is also involved in the progressive reduction of inflammation at the site of the wound by regulating the phenotypic transformation of macrophages into cells with an anti-inflammatory profile^3,7,17,18. 

In addition, a link has been shown between an increased expression of collagen III in the dermis and augmented regeneration potential following an injury. A recent study has shown that collagen III is an essential regulator of re-epithelialization, the activation of fibroblasts and of matrix architecture when skin wounds are healing. Collagen III promotes wound closure, thereby improving the efficacy and quality of the healing process¹⁹. By acting on the secretion of these biological markers, GM derived from Ceratonia siliqua improves the 4 biological events of the proliferation phase. 

The duration and severity of the inflammatory response affect healing quality. Although moderate inflammation favors wound repair, a more intense and chronic inflammatory phenomenon leads to a delay in healing, or even to the development of keloids or hypertrophic pathological scars^6,9,13. Macrophages play a fundamental role in each phase of healing because of their capacity to modulate the extent of inflammation in the tissue. They in fact exhibit different phenotypes, or polarization states, generally classified as pro-inflammatory (M1 macrophages) or anti-inflammatory (M2 macrophages). During the inflammatory phase occurring immediately after the wound, the phenotype of macrophages is primarily pro-inflammatory M1. M2 macrophages participate in the regeneration of damaged tissue by promoting angiogenesis and deposit of the extracellular matrix. It has been described that deregulating the polarization of macrophages can reduce healing quality. Indeed, larger quantities of the factors secreted by pro-inflammatory M1 macrophages were found in pathological scars^9,14,20. By acting on macrophages polarization and favoring the M2 phenotype, GM derived from Ceratonia siliqua reinforces their anti-inflammatory and pro-regenerating actions.

Re-epithelialization is an indispensable mechanism to restore an effective barrier function after an injury and, following its progression, is used as a parameter to define success of the healing process. Accelerating the reformation of a robust epidermal barrier is therefore a necessary prerequisite for any solution intended to repair wounded or damaged skin¹⁵. A kinetic study has revealed that GM derived from Ceratonia siliqua tested on wounded VI SILABSKIN FT accelerated wound closure (re-epithelialized surface: +34%) 30 hours after creating the wound and lead to the complete closure at t46h in comparison to the control. 

This active ingredient displays a double performance on repair dynamics by accelerating the rate of repair and improving the quality of repair. A clinical study was conducted on adult Caucasian volunteers with skin lesions resulting from a mechanical aggression that stripped the upper layers of the epidermis. This model is commonly used to assess the beneficial effects of wound healing treatments²¹. Formulated at 0.5% and 1% in a gel, GM derived from Ceratonia siliqua promoted recovery of the damaged cutaneous barrier as of the first day of application. This active ingredient acts promptly after application, thereby attenuating cutaneous erythema associated with skin lesion, with a significant improvement within two days of application with the formula at 1% and after four days with the formula at 0.5%. It also improves the quality of healing by significantly limiting the appearance of scabs and signs of skin drying.

Conclusion

This active ingredient is a novel solution for damaged skin in people of all ages. Resulting from six years of research, these galactomannans obtained from carob gum offer support to the healing process of wounded or damaged skin requiring repair. The ingredient is perfectly tolerated, and its therapeutic efficacy has been clinically demonstrated in a cohort of Caucasian adult subjects with skin lesions resulting from mechanical aggression. 

As of the first day of application of the product, the damaged barrier function is already significantly improved. As a result of this rapid effect on the barrier, it significantly attenuates erythema resulting from the injury and improves the quality of healing. This active ingredient accelerates healing and drying and scabs are reduced. It is an effective care product combining rapidity of action and efficacy in the care of wounded or damaged skin requiring repair.

References

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