Untersuchungen zur Immobilisierung und Freisetzung von rekombinantem humanem bone morphogenetic Protein 2 (BMP-2) in biologisch aktiver Form auf metallischen Implantatoberflächen

In den letzten Jahren wurde die chemische Immobilisierung von biologisch aktivem BMP-2 auf Titanimplantaten beschrieben, die zum besseren Knochenwachstum und zur beschleunigten Osteointegration in vivo führte [1-4]. Immobilisierungs- und Desorptionsexperimente wurden ebenfalls auf Cobaltchrommolybdän- und Titanplasmaspray-beschichteten Legierungsoberflächen durchgeführt. Zur Entwicklung eines solchen Systems soll die Freisetzung des immobilisierten BMP-2 kontrolliert werden. Wir verwenden je nach chemische Modifikation der Oberflächen zwei Methoden zur Immobilisierung: die kovalente und die nicht-kovalente. Für die nicht-kovalente Immobilisierung auf mit Chromschwefelsäure behandelten (CSA) Metalloberflächen wurde rhBMP-2 hydrophob auf alkylsubstituiertem Titan mit hoher Oberflächenkonzentration (Ti-CSA-APS) adsorbiert. Die kovalente Immobilisierung erfolgte durch Aktivierung der vorher mit 3-Aminopropyltriethoxysilan (APS) modifizierten Oberfläche mittels 1,1´- Carbonyldiimidazol (CDI) und anschließende Kopplung der ?-Aminogruppe der Lysinreste des Proteins. Im letzten Fall erhält man eine Mischung aus kovalent und nicht-kovalent immobilisiertem Protein. Die immobilisierten Mengen an 125I-rhBMP-2 auf Titan liegen je nach Oberflächenbeschaffenheit zwischen 0.2 8.0 µg/cm2 geometrischer Fläche. Die Freisetzungskinetiken von 125I-rhBMP-2 auf Titan und anderen Metalloberflächen liefern wichtige Information zur Verwendung der Metallimplantate als drug delivery in situ. Die Halbwertszeiten wurden nach einem Zweiphasen-Zerfallsmodell berechnet, wobei zwischen einer schnellen Anfangsphase und einer langsameren Endphase differenziert wird. Hier wird über die langsamere Phase berichtet. Im Falle einer spontanen Desorption unter Verwendung eines physiologischen Phosphat-Puffers bei pH 7.4 werden 90 % der ursprünglich immobilisierten Proteinmenge in der langsamen Phase mit Halbwertszeiten von 30 d für Ti-CSA-APS und 40 d für Ti-CSA-APS-CDI freigesetzt. Zur Differenzierung zwischen kovalent und nicht-kovalent gebundenem 125I-rhBMP-2 wurden Verdrängungsexperimente bei hohen Konzentrationen von Alkylaminlösungen durchgeführt. Zusätzlich dient die Desorption durch Alkylaminlösungen als Nachweis für den Mechanismus der Adsorption von BMP-2 durch hydrophobe Wechselwirkung. Letztere Methode führt zu einer schnellen Freisetzung von 30 % der ursprünglich immobilisierten Proteinmenge innerhalb von 6 h. Die restlichen 70 % werden mit Halbwertszeiten von 5 8 d von der Ti-CSA-APS- bzw. 13 d von der Ti-CSA-APS-CDI-Oberfläche freigesetzt. Im Gegensatz dazu, werden die Halbwertszeiten durch Ethylaminlösung auf 18 d für Ti-CSAAPS und 28 d für Ti-CSA-APS-CDI reduziert. Ähnliche Werte konnten für die modifizierte Cobaltchrommolybdän-Legierung erhalten werden. Daraus schließen wir einen Zusammenhang zwischen den Halbwertszeiten und der Kohlenstoffkettenlänge der verwendeten Alkylaminlösungen. Es wird eine stärkere hydrophobe Wechselwirkung bei hoher Kohlenstoffkettenlänge (C6, C7, C8) gezeigt. Die biologische Aktivität des auf Metalloberflächen immobilisierten rhBMP-2 konnte in der murinen Osteoblasten-Zelllinie MC3T3-M1 mittels Fluoreszenzmikroskopie [5] durch Aktivierung der alkalischen Phosphatase gemessen werden. Zellen, die auf rhBMP-2-beschichteten Metalloberflächen wachsen, zeigen nach 6 d im Gegensatz zu den Negativkontrollen eine starke Fluoreszenz als Nachweis der alkalischen Phosphatase-Aktivität. Diese Ergebnisse konnten in einem 1 mm-Spaltüberbrückungsverfahren in vivo im distalen Femurkondylus vom Schaf durch komplette Osteointegration nach 4 Wochen bestätigt werden. Die gemessenen Halbwertszeiten von 30 40 d stimmen sehr gut mit der Zeit überein, die für das physiologische Knochenwachstum erfordert wird. Nach Berechnungen von Desorptionsexperimenten von Titanplasmaspray-beschichteter Titanlegierung unter Berücksichtigung der Halbwertszeiten werden theoretisch ca. 80 ng/cm2 rhBMP-2 pro Tag freigesetzt. Das zeigt das Potential von bioaktiven Oberflächen für klinische Anwendungen

Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein 2 (rhBMP-2). A pilot study in dogs

The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm(2)), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm(2))] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependant manner

Poly(2-ethyl-2-oxazoline) grafted gellan gum for potential application in transmucosal drug delivery

Gellan gum (GG) has been used to prepare polymeric carriers with prolonged retention on the eye surface for topical ocular drug delivery. In this work, GG was chemically modified with short poly(2-ethyl-2-oxazoline) (PEtOx) chains that were expected to have minimal adhesion to mucosal tissues (mucoadhesion). The choice of synthetic procedure, solvents, and reagents has been dictated by biocompatibility of the materials and possible application in drug delivery. The grafts were synthesized via cationic ring-opening polymerization and their living chains were attached onto deprotonated gellan backbone. The derivatives with three degrees of grafting were prepared by varying the in-feed mass ratio of PEtOx grafts over GG. NMR and FT-IR spectroscopies, thermogravimetric analysis, and SEC evidenced that the grafting had actually taken place. However, a greater diffusion coefficient determined for the copolymer, using diffusion-ordered spectroscopy (NMR), in relation to the diffusion of the unmodified GG, suggested either partial degradation of the backbone or a more compact structure of the copolymer. GG and its graft copolymers (GG-g-PEtOx) were found to be highly biocompatible with cells cultured under their induction at concentration of 1, 0.1 and 0.01 mg/mL demonstrated a physiological morphology, as well as an increase in viability and proliferation.Peer reviewe

Design of a laboratory bioreactor for engineering articular cartilage based on 3D printed nasal septum-like scaffolds

«Η εκφύλιση των χόνδρων είναι μια σοβαρή πάθηση που επηρεάζει μεγάλο μέρος του πληθυσμού σε όλο το ηλικιακό φάσμα. Επί του παρόντος χρησιμοποιούνται διάφορες τεχνικές αποκατάστασης για μικρής έκτασης βλάβες όπως η αρθροπλαστική απόξεσης και ο υποχονδρικός τρυπανισμός, οι οποίες δεν μπορούν να επιδιορθώσουν βλάβες μεγαλύτερης έκτασης. Η Αναγεννητική Ιατρική προωθεί την Μηχανική Ιστών στο προσκήνιο των σύγχρονων μηχανικών τεχνικών, συνδυάζοντας καινοτόμα βιοσυμβατά υλικά, νέες μεθόδους μηχανικής ιστών όπως η τεχνολογία 3D εκτύπωσης και βιοδιαδικασίες που αποσκοπούν στην δημιουργία ποιοτικών μοσχευμάτων για εκτεταμένες βλάβες των χόνδρων. Κατάλληλο κυτταρικό περιβάλλον για δημιουργία ιστών μπορεί να επιτευχθεί με την ανάπτυξη αυτών των μοσχευμάτων σε βιοαντιδραστήρες. O κάθε βιοαντιδραστήρας χρησιμοποιεί διαφορετικές αρχές καλλιέργειας, και ορισμένοι από αυτούς όπως οι μικτού τύπου και οι βιοαντιδραστήρες διαπότισης επιστρατεύουν την άσκηση μηχανικών δυνάμεων επί του ικριώματος ώστε να επιτευχθεί μεγάλη κυτταρική πυκνότητα και ενισχυμένες μηχανικές ιδιότητες που οδηγούν στην δημιουργία καλύτερης ποιότητας χόνδρου. Αυτές οι ιδιαιτερότητες των βιοαντιδραστήρων μπορούν να αποτελέσουν εφαλτήριο κατασκευής εργαστηριακών βιοαντιδραστήρων, για την καλλιέργεια 3D εκτυπωμένων ρινικών διαφραγμάτων ως ένα λειτουργικό παράδειγμα υαλώδους χόνδρου».Cartilage degeneration is a severe disease affecting a significant part of the population at all ages. Various treatment modalities are currently used for small-sized cartilage defects, such as abrasion arthroplasty and subchondral drilling, but fail to repair larger-scale damages. Regenerative Medicine pushes Tissue Engineering (TE) to the forefront of modern engineering techniques combining novel biocompatible materials, new tissue engineering methods, like 3D printing technology and bioprocesses trying to create quality transplants for large cartilage defects. The appropriate cell environment for engineered tissues can be achieved through growth of the tissue-engineered constructs into bioreactors. Each bioreactor uses different principles for culturing processes, and some of them mostly mixed and perfusion bioreactors, use different kind of mechanical forces on the scaffold to achieve high cell densities, enhanced mechanical properties leading to better quality of engineered cartilage. These advantageous particularities can be used to create a laboratory bioreactor design, for culturing 3D printed nasal septum cartilage as a working example of hyaline cartilage

Translational research for nasal septum cartilage regeneration with chondrocytes derived from differentiated human adipose mesenchymal stem cells

Η εργασία αφορά στη μεταφραστική έρευνα ιστοτεχνολογίας και συγκεκριμένα στη δημιουργία ανθρώπινου ρινικού διαφράγματος με τη χρήση ηλεκτρονικά υποβοηθούμενου σχεδιασμού και τρισδιάστατης εκτύπωσης τρισδιάστατου (3D) πορώδους ικριώματος χιτοζάνης/ζελατίνης (CAD/CAM). Το ικρίωμα θα χρησιμοποιηθεί για να αποικιστεί από χρονδροκύτταρα που προκύπτουν από διαφοροποιημένα μεσεγχυματικά κύτταρα ανθρώπου προερχόμενα από λιπώδη ιστό (Adipose Tissue Mesenchymal Stem Cells-AD- MSCs). Η όλη διαδικασία επιτυγχάνεται με τη χρήση βιοαντιδραστήρα.Τα μεσεγχυματικά κύτταρα είναι πολυδύναμα βλαστοκύτταρα που μπορούν να απομονωθούν από το μυελό των οστώνκαι το λιπώδη ιστό. Τα κύτταρα αυτά έχουν τη δυνατότητα να διαφοροποιούνται, υπό εργαστηριακές συνθήκες, σε οστεοκύτταρα, χονδροκύτταρα, και λιποκύτταρα. Στην παρούσα μελέτη ανθρώπινα μεσεγχυματικά κύτταρα απομονώθηκαν από λιπώδη ιστό και καλλιεργήθηκαν in vitro. Η έκφραση των αντιγόνων επιφανείας CD90, CD73, σε συνδυασμό με την απουσία του μάρτυρα CD45 επιβεβαιώνουν την επιτυχή απομόνωση μεσεγχυματικών βλαστικών κυττάρων, με χρήση κυτταρομετρίας ροής. Έπειτα από 21 ημέρες από την επαγωγή στοχευόμενης διαφοροποίησης τα βλαστοκύτταρα διαφοροποιήθηκαν σε χονδροκύτταρα και χαρακτηρίστηκαν ιστολογικά με χρώση κυανού της τολουιδίνης και μοριακά με RTPCR για δείκτες διαφοροποίησης όπως η αγκρεκάνη. Με τη χρήση του τρισδιάστατου εκτυπωτή δημιουργήθηκε υπό κλίμακα ικρίωμα ρινικού χόνδρου από PLA. Η διαδικασία θα ολοκληρωθεί με την εκτύπωση του υπό διερεύνηση υλικού χιτοζάνης/ζελατίνης σε 3D ικρίωμα και αφού εμποτιστεί με χονδροκύτταρα θα μεταφερθεί στον βιοαντιδραστήρα.Mesenchymal stem cells (MSCs) are multipotent cells isolated from various tissues, mainly from the bone marrow and adipose tissue. Their ability to differentiate into osteoblasts, chondrocytes or adipocytes renders them a promising clinical tool for injury repair and tissue regeneration. In the current study, MSCs were isolated from human adipose tissue (hAD-MSCs) and were triggered to differentiate into chondrocytes in vitro. Expression of mesenchymal stem cell markers, such as CD90 and CD73, in combination with the absence of hematopoietic markers, such as CD45, proves via flow cytometry the successful isolation of MSCs. Histologic staining with Toluidine blue and real time PCR analysis for the expression of the chondrogenic marker aggrecan (ACAN) verified the successful chondrogenic differentiation of AD-MSCs. Using Poly Lactic-Acid as scaffolding material, a three-dimensional scaffold with customized architecture, controlled porosity and interconnected porous structure was fabricated using 3D printing. The produced scaffold represents the morphology of the nasal septum cartilage. We aspire, to see this scaffold with the differentiated chondrocytes and culture the complex under the appropriate micoenvironmental conditions of a bioreactor system in order to regenerate a potential cartilage transplant. This in vitro study expands the potentials of human AD-MSCs to be used in clinic for alleviation of cartilage defects and tissue engineering in Greece and worldwide

A computational analysis of a novel therapeutic approach combining an advanced medicinal therapeutic device and a fracture fixation assembly for the treatment of osteoporotic fractures: Effects of physiological loading, interface conditions, and fracture fixation materials

: The occurrence of periprosthetic femoral fractures (PFF) has increased in people with osteoporosis due to decreased bone density, poor bone quality, and stress shielding from prosthetic implants. PFF treatment in the elderly is a genuine concern for orthopaedic surgeons as no effective solution currently exists. Therefore, the goal of this study was to determine whether the design of a novel advanced medicinal therapeutic device (AMTD) manufactured from a polymeric blend in combination with a fracture fixation plate in the femur is capable of withstanding physiological loads without failure during the bone regenerative process. This was achieved by developing a finite element (FE) model of the AMTD together with a fracture fixation assembly, and a femur with an implanted femoral stem. The response of both normal and osteoporotic bone was investigated by implementing their respective material properties in the model. Physiological loading simulating the peak load during standing, walking, and stair climbing was investigated. The results showed that the fixation assembly was the prime load bearing component for this configuration of devices. Within the fixation assembly, the bone screws were found to have the highest stresses in the fixation assembly for all the loading conditions. Whereas the stresses within the AMTD were significantly below the maximum yield strength of the device's polymeric blend material. Furthermore, this study also investigated the performance of different fixation assembly materials and found Ti-6Al-4V to be the optimal material choice from those included in this study

Effect of Uniaxial Compression Frequency on Osteogenic Cell Responses in Dynamic 3D Cultures

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 μm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity

The enrichment of whey protein isolate hydrogels with poly-γ-glutamic acid promotes the proliferation and osteogenic differentiation of preosteoblasts

© 2023 The authors. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3390/gels10010018Osseous disease accounts for over half of chronic pathologies, but there is a limited supply of autografts, the gold standard; hence, there is a demand for new synthetic biomaterials. Herein, we present the use of a promising, new dairy-derived biomaterial: whey protein isolate (WPI) in the form of hydrogels, modified with the addition of different concentrations of the biotechnologically produced protein-like polymeric substance poly-γ-glutamic acid (γ-PGA) as a potential scaffold for tissue regeneration. Raman spectroscopic analysis demonstrated the successful creation of WPI-γ-PGA hydrogels. A cytotoxicity assessment using preosteoblastic cells demonstrated that the hydrogels were noncytotoxic and supported cell proliferation from day 3 to 14. All γ-PGA-containing scaffold compositions strongly promoted cell attachment and the formation of dense interconnected cell layers. Cell viability was significantly increased on γ-PGA-containing scaffolds on day 14 compared to WPI control scaffolds. Significantly, the cells showed markers of osteogenic differentiation; they synthesised increasing amounts of collagen over time, and cells showed significantly enhanced alkaline phosphatase activity at day 7 and higher levels of calcium for matrix mineralization at days 14 and 21 on the γ-PGA-containing scaffolds. These results demonstrated the potential of WPI-γ-PGA hydrogels as scaffolds for bone regeneration.Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.Published onlin