Injectable hyaluronic acid based hydrogels for the repair of cartilage lesions

Hyaluronic acid (HA) is a natural polysaccharide which is found natively in cartilage tissue. HA hydrogels are formed by the modification of HA through its carboxyl and hydroxyl groups and subsequent crosslinking. For applications in cartilage repair, it has been found that HA hydrogels not only support and maintain chondrocyte viability and phenotype when cultured in vitro and in vivo, but also that HA hydrogel chemistry supports and promotes the chondrogenic differentiation of mesenchymal stem cells (MSCs). A promising, non-invasive method for the repair of cartilage lesions is based on the use of injectable hydrogels with desirable properties in combination with biomolecules and cells. Please click Additional Files below to see the full abstract

Biomimetic Cell-Laden MeHA Hydrogels for the Regeneration of Cartilage Tissue

Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA (CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology

Injectable hyaluronic acid based hydrogels for articular cartilage repair

The purpose of this thesis is to develop injectable hydrogels based on hyaluronic acid, a natural polymer found in the human body, for the repair of cartilage lesions. The existing procedures for the repair of cartilage lesions are invasive and have limitations with respect to geometry and cartilage defect size. Therefore, there is a great need for the development of non-invasive treatments for the repair of small cartilage injuries. In this context, the development of injectable methacrylated hyaluronic acid (MeHA) hydrogels, synthesized using two different crosslinking methods, has been effected. In particular, a redox initiation system of ammonium persulfate (APS) and N,N,N,N΄- tetramethylenediamine (TEMED), and a matrix metalloproteinase 7 (MMP7)-sensitive peptide were effectively used for the formation of MeHA based hydrogels. During this study, the influence of the molecular weight of HA, degree of methacrylation (DM), and biofunctionalization of MeHA using a chondroitin sulfate (CS) binding peptide, was experimentally assessed. Concerning the use of the MMP7-degradable peptide as crosslinker, it was observed that, as the concentration of the peptide crosslinker increased, the gelation onset time and the degradation rate of the synthesized hydrogels were decreased, while their storage modulus (G′) increased. The use of CS-MeHA as a functional macromer resulted in a retardation of the gelation onset and the formation of softer hydrogels. Finally, the crosslinking reaction in cell growth medium was shown to increase the G’ of the formed hydrogel to values that could favor the differentiation of human mesenchymal stem cells (hMSCs) to chondrocytes. The successful development of injectable MeHA hydrogels using an MMP7-degradable peptide as crosslinker was followed by their embedding with a low concentration of either human mesenchymal stem cells (hMSCs) or chondrocytes. hMSC-laden MeHA and CS-MeHA hydrogels were cultured either in stem cell medium (SCM) or chondrogenic medium (CiM), and assessed regarding their ability to promote the differentiation of hMSCs towards a chondrogenic and/or hypertrophic phenotype, as well as to maintain the viability of the encapsulated cells. The developed MeHA and CS-MeHA hydrogels appeared to provide a proper environment for the growth and proliferation of hMSCs. This was enhanced in the case of CS-MeHA hydrogels showing the positive effect of the chondroitin sulfate binding peptide on the chondrogenic differentiation of hMSCs. However, the use of chondrogenic medium was considered necessary in order to obtain full-span chondrogenesis. Finally, the chondrocyte-laden MeHA hydrogels were cultured in an ex vivo osteochondral platform and assessed regarding their ability to form cartilage extracellular matrix. Distribution of glycosaminoglycans and development of chondrocyte clusters in isogenous groups, characteristic of hyaline cartilage morphology, were observed on the fourteenth day of the ex vivo culture.Σκοπός της παρούσας διατριβής είναι η ανάπτυξη ενέσιμων υδροπηκτών με βάση το υαλουρονικό οξύ για την επιδιόρθωση χόνδρινων ατελειών. Οι ήδη υπάρχουσες τεχνικές για τη θεραπεία χόνδρινων ατελειών είναι επεμβατικές και παρουσιάζουν περιορισμούς σχετικά με τη γεωμετρία και το μέγεθος του τραύματος. Επομένως, υπάρχει μεγάλη ανάγκη για την ανάπτυξη μη επεμβατικών θεραπειών με βάση τις αρχές της μηχανικής ιστών. Βάσει των παραπάνω, μελετήθηκε η ανάπτυξη ενέσιμων υδροπηκτών μεθακρυλιωμένου υαλουρονικού οξέος (MeHA), που συντέθηκαν με δύο διαφορετικές μεθόδους δικτύωσης. Πιο συγκεριμένα, ένα σύστημα εκκινητών οξείδωσης-αναγωγής υπερθειικού αμμωνίου και Ν,Ν,Ν,Ν’-τετραμεθυλοδιαμίνης και ένα πεπτίδιο που διασπάται ενζυμικά από τη μεταλλοπρωτεϊνάση θεμέλιας ουσίας 7 χρησιμοποιήθηκαν για τον σχηματισμό υδροπηκτών με βάση το μεθακρυλιωμένο υαλουρονικό οξύ. Κατά τη διάρκεια της παρούσας μελέτης αξιολογήθηκε πειραματικά η επίδραση του μοριακού βάρους του υαλουρονικού οξέος, του βαθμού μεθακρυλίωσης, και της τροποποίησης του μεθακρυλιωμένου υαλουρονικού οξέος χρησιμοποιώντας ένα πεπτίδιο προσκόλλησης θειικής χονδροϊτίνης (CS), ως προς τις ιδιότητες των υδροπηκτών. Όσον αφορά τη χρήση του πεπτιδίου που διασπάται ενζυμικά από τη μεταλλοπρωτεϊνάση θεμέλιας ουσίας 7 ως μέσο δικτύωσης, παρατηρήθηκε ότι με την αύξηση της συγκέντρωσης του πεπτιδίου, ο χρόνος έναρξης σχηματισμού της υδροπηκτής και ο ρυθμός διάσπασης των συντιθεμένων υδροπηκτών μειώνονται, ενώ το μέτρο αποθήκευσής τους (G’) αυξάνεται. Η χρήση του CS-MeHA ως λειτουργικό μακρομερές είχε ως αποτέλεσμα την καθυστέρηση της έναρξης σχηματισμού των υδροπηκτών και τη δημιουργία μαλακών υδροπηκτών. Τέλος, η αντίδραση δικτύωσης σε θρεπτικό μέσο ανάπτυξης κυττάρων έδειξε ότι το G’ της σχηματιζόμενης υδροπηκτής αυξάνεται σε βαθμό που θα μπορούσε να ευνοήσει τη διαφοροποίηση ανθρώπινων μεσεγχυματικών βλαστικών κυττάρων (hMSCs) σε χονδροκύτταρα. Την επιτυχή ανάπτυξη ενέσιμων υδροπηκτών με χρήση πεπτιδίου που διασπάται ενζυμικά από τη μεταλλοπρωτεϊνάση θεμέλιας ουσίας 7 ως μέσο δικτύωσης, ακολούθησε η ενσωμάτωση σε αυτές ανθρώπινων μεσεγχυματικών βλαστικών κυττάρων, ή χονδροκυττάρων, σε χαμηλή συγκέντρωση. Οι υδροπηκτές MeHA και CS-MeHA, στις οποίες ενσωματώθηκαν hMSCs, καλλιεργήθηκαν είτε σε θρεπτικό μέσο βλαστοκυττάρων είτε σε χονδρογονικό μέσο και εκτιμήθηκαν σχετικά με την ικανότητά τους να προάγουν τη διαφοροποίηση των hMSCs ως προς έναν χονδρογονικό και/ή υπερτροφικό φαινότυπο, καθώς και ως προς την ικανότητά τους να διατηρούν τη βιωσιμότητα των ενσωματωμένων κυττάρων. Παρατηρήθηκε ότι οι υδροπηκτές MeHA και CS-MeHA παρέχουν ένα κατάλληλο περιβάλλον για την ανάπτυξη και τον πολλαπλασιασμό των hMSCs. Αύξηση των χονδρογονικών δεικτών παρατηρήθηκε και στην περίπτωση των υδροπηκτών CS-MeHA υποδεικνύοντας τη θετική επίδραση του πεπτιδίου προσκόλλησης θειικής χονδροϊτίνης στη χονδρογονική διαφοροποίηση των hMSCs. Ωστόσο, η χρήση χονδρογονικού μέσου κρίθηκε απαραίτητη για την επίτευξη χονδρογένεσης πλήρους εύρους. Τέλος, υδροπηκτές MeHA, στις οποίες ενσωματώθηκαν χονδροκύτταρα, καλλιεργήθηκαν σε μια ex vivo οστεοχόνδρινη πλατφόρμα και αξιολογήθηκαν σχετικά με την ικανότητά τους να σχηματίζουν χόνδρινη εξωκυττάρια θεμέλια ουσία. Κατά την δέκατη τέταρτη ημέρα της ex vivo καλλιέργειας παρατηρήθηκε κατανομή γλυκοζαμινογλυκανών και ανάπτυξη συστάδων χονδροκυττάρων σε ισογενείς ομάδες τα οποία αποτελούν χαρακτηριστικό της μορφολογίας του υαλώδους χόνδρου

Recent Developments in 3D-(Bio)printed Hydrogels as Wound Dressings

Wound healing is a physiological process occurring after the onset of a skin lesion aiming to reconstruct the dermal barrier between the external environment and the body. Depending on the nature and duration of the healing process, wounds are classified as acute (e.g., trauma, surgical wounds) and chronic (e.g., diabetic ulcers) wounds. The latter take several months to heal or do not heal (non-healing chronic wounds), are usually prone to microbial infection and represent an important source of morbidity since they affect millions of people worldwide. Typical wound treatments comprise surgical (e.g., debridement, skin grafts/flaps) and non-surgical (e.g., topical formulations, wound dressings) methods. Modern experimental approaches include among others three dimensional (3D)-(bio)printed wound dressings. The present paper reviews recently developed 3D (bio)printed hydrogels for wound healing applications, especially focusing on the results of their in vitro and in vivo assessment. The advanced hydrogel constructs were printed using different types of bioinks (e.g., natural and/or synthetic polymers and their mixtures with biological materials) and printing methods (e.g., extrusion, digital light processing, coaxial microfluidic bioprinting, etc.) and incorporated various bioactive agents (e.g., growth factors, antibiotics, antibacterial agents, nanoparticles, etc.) and/or cells (e.g., dermal fibroblasts, keratinocytes, mesenchymal stem cells, endothelial cells, etc.)

Recent Developments in Hyaluronic Acid-Based Hydrogels for Cartilage Tissue Engineering Applications

Articular cartilage lesions resulting from injurious impact, recurring loading, joint malalignment, etc., are very common and encompass the risk of evolving to serious cartilage diseases such as osteoarthritis. To date, cartilage injuries are typically treated via operative procedures such as autologous chondrocyte implantation (ACI), matrix-associated autologous chondrocyte implantation (MACI) and microfracture, which are characterized by low patient compliance. Accordingly, cartilage tissue engineering (CTE) has received a lot of interest. Cell-laden hydrogels are favorable candidates for cartilage repair since they resemble the native tissue environment and promote the formation of extracellular matrix. Various types of hydrogels have been developed so far for CTE applications based on both natural and synthetic biomaterials. Among these materials, hyaluronic acid (HA), a principal component of the cartilage tissue which can be easily modified and biofunctionalized, has been favored for the development of hydrogels since it interacts with cell surface receptors, supports the growth of chondrocytes and promotes the differentiation of mesenchymal stem cells to chondrocytes. The present work reviews the various types of HA-based hydrogels (e.g., in situ forming hydrogels, cryogels, microgels and three-dimensional (3D)-bioprinted hydrogel constructs) that have been used for cartilage repair, specially focusing on the results of their preclinical and clinical assessment

Immunoinformatics Approach to Design a Multi-Epitope Nanovaccine against <i>Leishmania</i> Parasite: Elicitation of Cellular Immune Responses

Leishmaniasis is a vector-borne disease caused by an intracellular parasite of the genus Leishmania with different clinical manifestations that affect millions of people worldwide, while the visceral form may be fatal if left untreated. Since the available chemotherapeutic agents are not satisfactory, vaccination emerges as the most promising strategy for confronting leishmaniasis. In the present study, a reverse vaccinology approach was adopted to design a pipeline starting from proteome analysis of three different Leishmania species and ending with the selection of a pool of MHCI- and MHCII-binding epitopes. Epitopes from five parasite proteins were retrieved and fused to construct a multi-epitope chimeric protein, named LeishChim. Immunoinformatics analyses indicated that LeishChim was a stable, non-allergenic and immunogenic protein that could bind strongly onto MHCI and MHCII molecules, suggesting it as a potentially safe and effective vaccine candidate. Preclinical evaluation validated the in silico prediction, since the LeishChim protein, encapsulated simultaneously with monophosphoryl lipid A (MPLA) into poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles, elicited specific cellular immune responses when administered to BALB/c mice. These were characterized by the development of memory CD4+ T cells, as well as IFNγ- and TNFα-producing CD4+ and CD8+ T cells, supporting the potential of LeishChim as a vaccine candidate

Biomimetic cell-laden meha hydrogels for the regeneration of cartilage tissue

Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA(CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology

Biomimetic Cell-Laden MeHA Hydrogels for the Regeneration of Cartilage Tissue

Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA(CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology