Development of hyaluronic acid/plasma derived fibrin hydrogels for the optimization of dermo-epidermal skin substitutes
- Autores: Andrés Montero Simón
- Directores de la Tesis: Diego Velasco Bayón (dir. tes.), José L. Jorcano Noval (codir. tes.)
- Lectura: En la Universidad Carlos III de Madrid ( España ) en 2019
- Idioma: español
- Tribunal Calificador de la Tesis: José Pérez Rigueiro (presid.), Lucía Martínez Galdeano (secret.), Abhay Pandit (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencia e Ingeniería de Materiales por la Universidad Carlos III de Madrid
- Materias:
- Enlaces
- Tesis en acceso abierto en: e-Archivo
- Resumen
Skin is the outermost organ of the body and covers almost its entire surface. Skin main functions are serving as protection against the hostile environment, regulating the body temperature, inhibiting water loss and also functioning as sensory organ. Skin damage thus is presented as a potential threat to the maintenance of the homeostasis between the body and its surroundings. Full-thickness wounds resulting from acute damage, severe burns and other chronic conditions like diabetic foot ulcer represent a huge medical burden due to their difficult treatment and high cost, fact that will make the skin wound care market to reach a $12.45 billion dollars market capitalization by 2022. Classical solutions to skin damage have been the use of autografts and allografts to transplant healthy skin parts into the wounded area. However, drawbacks associated to these techniques have rendered them of limited utility and the emergence of skin tissue engineering and more precisely of dermo-epidermal constructs has opened the possibility to fully treat these wounds in an effective manner.
In this thesis, we use a solution previously developed in our laboratory consisting on a dermo-epidermal skin equivalent comprised of a plasma-derived fibrin dermal scaffold containing embedded human primary fibroblast and a layer of keratinocytes seeded on top to form the epidermis. Although this matrix has been proven to be effective for the treatment of severe and extensive burned patients in Spain, several persisting issues associated with this solution still remain unsolved. Here we perform a complete characterisation of the plasma-derived matrices and the effects of different environments on them in terms of contractile behaviour and mechanical endurance, to later propose a solution to the issues related to them in the form of poor mechanical properties and excessive degradation rates.
The first chapter of this thesis comprises a deep review of the concepts necessary to completely understand this thesis including background from the fields of biomaterials and tissue engineering. This introduction to the topic explores the concepts of skin, skin damage, tissue engineering, hydrogels, human plasma-derived hydrogels and hyaluronic acid which will serve the reader to further understand the rationale behind the experiments carried out in this thesis.
The second chapter of the thesis describes the experiments performed to determine the optimal final fibrin concentration to generate the dermo-epidermal equivalents in terms of fast gelation times, low degradation rates and good mechanical properties. Two different final concentrations were proposed, 1.2 mg/ml and 2.4 mg/ml with the information we had from previous studies. Matrices used for burnt patients had classically used the lower concentration due to efficiency reasons since the fibrin is obtained from the patient blood and their condition combined with great amount of surface damaged impedes the extraction of high amounts of fluid. With this premise, increasing the concentration of fibrin was first evaluated as a solution to overcome the persisting poor mechanical properties reported by clinicians.
The rationale behind the performance of a series of experiments was to evaluate the suitability of the fibrin concentration to generate a matrix where cells can proliferate, migrate and remodel the structure. In order to do so, gelation time and kinetics, contractile behaviour in the presence and absence of cells, internal structure, protein release, mechanical properties and human primary fibroblast and keratinocytes viability were evaluated.
The ability of hydrogels to uptake and release great amount of water is closely related with the presence and the balance of hydrophilic and hydrophobic groups in their structure [1]. The swelling behaviour of the two systems when submerged in PBS at 37oC was measured and no differences in terms of weight changes were recorded for the fibrin hydrogels at 1.2 mg/ml and 2.4 mg/ml of concentration. An overall 25% mass loss was found after the first 24 hours, with no further weight decrease for the next time points (up to 10 days). Parallelly, the release of blood plasma proteins to the environment was measured yielding as result the expected double amount of proteins escaping the 2.4 mg/ml fibrin hydrogels as it contained double the amount of initial plasma.
Mechanically, as expected, doubling the final concentration doubled the Young’s modulus of the specimens in the compressive test, result similar to that reported on the effect of fibrin and thrombin concentration in the stiffness of the hydrogels [2]. Further characterisation of the polymeric matrices was assessed through the internal visualization of the hydrogels with SEM. Difference in the structural nature of the two concentrations evidenced the differences found in mechanical properties. Hydrogels at 1.2 mg/ml final fibrin concentration were found to be more porous and thus mechanically less stable.
Fibroblasts behaviour inside the matrices was correlated in terms of capability to contract the fibrin hydrogels and in terms of proliferation. The attachment of human primary fibroblast to the matrix through the RGD cell-adhesion motifs present in the matrix was confirmed as the hydrogels tend to contract at a bigger pace than in the absence of cells [3]. Cell-mediated contraction and proliferation were significantly decreased with hydrogels at 2.4 mg/ml with respect to the lower concentrated 1.2 mg/ml fibrin. This behaviour is in line with previous reports on the lower proliferation of cells with the increasing fibrin concentration due to a more compact matrix delaying the remodelling by the fibroblasts [2,4]. Finally, human primary keratinocytes proliferation when seeded on top of the matrices showed a significant decrease in activity through the MTS assay for the hydrogels at 2.4 mg/ml fibrin concentration. This result evidences that stiffer surfaces sensed by the cells are slowing the rate of proliferation for our system and thus are making the use softer matrices the best choice for the generation of the bioengineered skin.
In this chapter 1.2 mg/ml hydrogels have shown worse mechanical properties and cell-mediated contraction rates, these negative results have been outweighed by their better or equal performance in terms of cell-free contraction and most importantly human primary fibroblast and keratinocytes proliferation and viability, the key aspects for the correct wound healing of damaged skin [5]. In that sense, 1.2 mg/ml fibrin concentration hydrogels were selected to generate our dermo-epidermal equivalents and to enhance their performance through the incorporation of other biomaterial, the hyaluronic acid (HA).
Chapter 3 of this thesis have been devoted to the study of the contraction of the 1.2 mg/ml final fibrin concentration matrices when subjected to different temperatures. The characterisation of the behaviour of the matrix under different conditions allowed us to better understand the processed occurring in the hydrogels and better design a strategy to reduce the detrimental behaviour of the equivalents. Three different temperatures (4ºC, 23ºC and 37ºC) were used to characterise the contractile behaviour of the plasma-derived fibrin matrices and commercial fibrinogen hydrogels were used as control to try to discern the origin of the shrinkage of the matrices.
The results obtained for the contraction of both human plasma and commercial fibrinogen hydrogels show that all matrices contract after incubation in an aqueous environment regardless of the temperature. Plasma hydrogels show a bi-phasic deswelling behaviour with an initial quick loss of mass up to 24 hours. This contraction is explained by the intrinsic nature of the fibrin hydrogels to deswell in aqueous solution. However, the lower loss of mass reported for plasma hydrogels compared to commercial fibrinogen is due to the presence of proteins and lipids in their structure, that are capable of modulating the contraction. Both the release of proteins and the bi-phasic behaviour of hydrogels previously reported by Zhang et al. for collagen hydrogels [6], showing an outer hydrophilic part and an inner hydrophobic part, could be the explanation for the contraction of the matrices. The contraction of fibrin matrices might be related with the aggregation of intermolecular assemblies when the hydrogels are submerged in aqueous solution. In this environment the non-polar groups attract to each other forming new crosslinking points that make the scaffold to be less porous and thus release more water to the environment, causing a mass decrease [6].
The rheological study performed at the three defined temperatures showed the absence of what is known as the strain hardening effect. This effect is defined as the increase in elastic modulus (G’) with the increasing values of strain amplitude and has been well characterised in commercial fibrinogen hydrogels [7,8]. The presence of this effect in commercial fibrinogen structures is due to the flexible nature of the fibres in line with the polymer network theory of flexible chains that define the hardening of the fibres upon the application of increasing strains to the polymer network [9,10]. In the case of plasma hydrogels, this effect was not found as evidenced in the plateau found in elastic modulus with the increasing strain amplitude when performing the strain sweep experiment. As previously reported by Shah et al. the elimination of this effect in plasma-derived hydrogels might be due to the presence of proteins and platelets in the structure [8].
In this chapter we have thoroughly studied the fibrin matrix and its behaviour under different environments in terms of temperature. This is an important characterisation of the material before continuing to add a supplementary biomaterial (hyaluronic acid) in order to reinforce the matrix and enhance the biological properties.
In chapter 4, natural HA purchased from Lifecore Biomedical was incorporated into the plasma-derived fibrin matrices in order to enhance the mechanical properties and better mimic the ECM formed upon skin damage. HA interacts with fibrinogen in a specific and reversible manner to form reinforced hydrogels with potential capability to serve as regeneration aid in skin damage [11]. Levels of HA increase upon tissue damage, evidencing its role in the wound healing process as explained in the introduction to this thesis [12]. However, the effect of HA in the fibrin matrix needs to be explored in order to render these hybrid matrices useful for skin tissue engineering.
In this chapter, we successfully incorporated the natural HA into the plasma-derived matrices. The results for the hydrogel contraction in the absence of human primary fibroblast show that increasing the concentration of natural HA incorporated increased the mass contraction of hydrogels with time, an undesired outcome due to the loss of material too quickly leading to failure of the hydrogels mechanically. The probable reason behind this behaviour is the weak interaction between HA and fibrin making that the material incorporated can potentially scape easily from the disrupted fibrin matrix and thus result in an overall loss of mass with incubation time, although it has not been reported before.
Mechanical properties results evidenced the low compressive modulus of elasticity for the hydrogels that contained natural HA. These low mechanical performance was expected after the results obtained in the swelling study, with high water loss ratios making the hydrogels less stable in vitro. The final study performed was the human primary fibroblast proliferation assay inside the matrices. Results show an increase in metabolic activity of cells as the amount of HA increased from 0.05% to 0.2%, with this last final concentration of natural HA obtaining better proliferation that the plasma control. Although the performance of cell proliferation inside the HA-Fibrin matrices was shown to be equal or even slightly higher than plasma alone, the poor results obtained for the rest of the experiments and the main driver of this thesis of improving the matrices mechanically lead us to try to find an alternative reinforcement material to enhance the performance of human plasma-derived matrices for skin tissue engineering. In that context, the possibility of using a chemically modified HA that could be crosslinked and thus could better stabilise the plasma matrix was explored.
Chapter 5 explored the the incorporation of HA-PEGDA into the plasma-derived fibrin hydrogels and gelation time and kinetics, contraction both with and without cells, protein release, HA release from the matrices, mechanical and structural characterisation, cellular (fibroblast and keratinocytes) proliferation in the presence of HA-PEGDA and finally generation of organotipic skin constructs in vitro were performed.
The incorporation of a new polymer network inside plasma hydrogels consisting of a commercially modified thiolated HA and PEGDA crosslinker was demonstrated in this work. This achievement opens the possibility of incorporating multiple polymer of different nature in order to enhance the performance of dermo-epidermal equivalents derived from human plasma. As explained in the introduction, the crosslinking occurs by means of the simple click chemistry, capable of yielding high efficiency in the reaction with no unreactive species generating side products and under mild conditions compatible with biological processes [13].
In this chapter, the addition of three concentrations of HA (0.05%, 0.1% and 0.2% w/v) with four levels of PEGDA crosslinking (2:1, 6:1, 10:1 and 14:1) have been evaluated through a series of experiments comprising both the material and biological properties of the newly formed hydrogels.
The swelling behaviour of the plasma-derived fibrin hydrogels incorporated with thiolated HA and PEGDA, showed that a decrease in the contraction behaviour ranging from a mere 5% to a whopping 25% occurred for all the conditions tested. The study of the mass loss of hydrogels is of vital importance in the clinical application, as an excessive contraction would lead to a failure in the transplant or in the development of scar tissue or even fibrotic scarring [14,15]. The swelling behaviour is based on the presence of hydrophilic groups in the composition of the hydrogel (-COOH; −SO3H; -OH; -CONH) [16] and on the environmental conditions such as temperature and pH that can greatly influence the behaviour of hydrogels [1]. In the case of thiolated HA, the presence in its structure of polar groups (-OH; -COOH and –SH) might influence the swelling behaviour by retaining more water inside the structure than the control plasma hydrogels.
Mechanical properties of the scaffolds play a crucial role in the correct development of engineered tissue both in vitro and in vivo. Skin has been previously characterised mechanically, yielding torsion strength values ranging from 0.4 to 0.85 MPa for the dermal component [17]. A majority of current scaffolds designed to bear cells inside have been reported to have very poor mechanical properties leading to early stage failures in the development of engineered tissues [18]. In this context, the incorporation of thiolated HA crosslinked with PEGDA has demonstrated an increase in stiffness of the hydrogels with respect to the plasma control. The natural stability of the HA- PEGDA hydrogels comes from the bonds formed through thiol-ene chemistry and the different levels of crosslinking, that have been demonstrated to be able to tune the mechanical properties of the hydrogels [19]. When HA-PEGDA was incorporated into the plasma-derived fibrin hydrogels, an increase in the Young’s modulus evidenced the increase in stiffness for these types of modified gels with the increasing amount of HA concentration (0.05%, 0.1% or 0.2% w/v).
The results evidence that the increase in rigidity of the matrices when the HA- PEGDA is incorporated results in a decrease in contraction in the presence of primary fibroblast. Other factor that might me affecting the contraction of the matrices is the correct proliferation and viability of the fibroblast inside the hydrogels containing thiolated HA and PEGDA crosslinker. As shown in the results, an overall decrease in proliferation was found for all hydrogels containing any concentration of HA and level of crosslinker after 7 days with respect to the plasma control. This alone is evidencing that fibroblast are finding difficulties to spread inside the more compact matrices and remodelling is taking longer than usual [20]. This effect was further evidence by the finding that fibroblasts encapsulated inside the hydrogels at 0.2% HA and 2:1 PEGDA crosslinking were still in a round morphology after 48 hours of incubation in the Live/Dead assay performed. Fibroblast proliferation activity after 14 days gave us evidence that the type of reaction taking place (thiol-ene chemistry) is suitable for biological components and does not elicit a detrimental reaction for cells [19].
A part from fibroblasts encapsulated inside the matrices, a second type of cellular component is needed for the generation of organotypic skins in vitro. Human primary keratinocytes were evaluated in the presence of thiolated HA and PEGDA crosslinker in order to assess their proliferative ability with respect to the plasma-derived matrices used as control. The results in this case show a decrease in the metabolic activity of keratinocytes when they are feeling the more hydrated surface of the matrix when high amounts of HA and PEGDA crosslinker are incorporated. This effect caused a decrease in the differentiation rate and thus the incorrect formation of skin tissue in the organotypic skin generation experiment. The more hydrated surface was detrimental as it maintained the surface of the keratinocyte layer with liquid impeding the complete drying of the layer and thus probably inhibiting the correct differentiation into the different strata that conform the epidermis.
The histology results showed that the incorporation of a mild amount of HA (0.05%) crosslinked at the highest ratio (2:1) was the optimal condition to generate fully differentiated skins while enhancing the mechanical properties of the constructs. Expression of the most common markers of skin (Vimentin, K5, K10 and Filaggrin) was found in the dermo-epidermal equivalents generated with HA crosslinked with PEGDA. Compared to the plasma controls, K10 showed a more confined and homogeneous expression in the suprabasal layer of the epidermis, showing thus a better final skin. Furthermore, dermis was shown to be up to 3 times thicker compared to the plasma control and the formation of a stratum corneum was enhanced for the constructs containing HA crosslinked with PEGDA as evidenced in the filaggrin expression.
This thesis has served as proof that the optimisation of human plasma-derived hydrogels for skin tissue engineering is achievable and that there is always room for improvements to be found in the form of new combination of materials that can better mimic the in vivo conditions found in nature. Better resemblance of these tissues will lead to cells behaving in a more similar manner as they do in their natural environment, opening the possibility to explore factors and cell types that completely regenerate tissues.
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