The Degradation of Synthetic Polymeric Scaffolds With Strut-like Architecture Influences the Mechanics-dependent Repair Process of an Osteochondral Defect in Silico

Current clinical treatments of osteochondral defects in articulating joints are frequently not successful in restoring articular surfaces. Novel scaffold-based tissue engineering strategies may help to improve current treatment options and foster a true regeneration of articulating structures. A frequently desired property of scaffolds is their ability to degrade over time and allow a full restoration of tissue and function. However, it remains largely unknown how scaffold degradation influences the mechanical stability of the tissue in a defect region and, in turn, the regenerative process. Such differing goals-supporting regeneration by degrading its own structure-can hardly be analyzed for tissue engineered constructs in clinical trials and in vivo preclinical experiments. Using an in silico analysis, we investigated the degradation-induced modifications in material and architectural properties of a scaffold with strut-like architecture over the healing course and their influence on the mechanics-dependent tissue formation in osteochondral defects. The repair outcome greatly varied depending on the degradation modality, i.e. surface erosion or bulk degradation with and without autocatalysis, and of the degradation speed, i.e. faster, equal or slower than the expected repair time. Bulk degradation with autocatalysis, independently of degradation speed, caused the mechanical failure of the scaffold prior to osteochondral defect repair and was thereby deemed inappropriate for further application. On the other hand, scaffolds with strut-like architecture degrading by both surface erosion and bulk degradation with slow degradation speed resulted in comparably good repair outcomes, thereby indicating such degradation modalities as favorable for the application in osteochondral defects

Scaffold-Dependent Mechanical and Architectural Cues Guide Osteochondral Defect Healing in silico

Osteochondral defects in joints require surgical intervention to relieve pain and restore function. However, no current treatment enables a complete reconstitution of the articular surface. It is known that both mechanical and biological factors play a key role on osteochondral defect healing, however the underlying principles and how they can be used in the design of treatment strategies remain largely unknown. To unravel the underlying principles of mechanobiology in osteochondral defect healing, i.e., how mechanical stimuli can guide biological tissue formation, we employed a computational approach investigating the scaffold-associated mechanical and architectural properties that would enable a guided defect healing. A previous computer model of the knee joint was further developed to simulate healing of an empty osteochondral defect. Then, scaffolds were implanted in the defect and their architectures and material properties were systematically varied to identify their relevance in osteochondral defect healing. Scaffold mechanical and architectural properties were capable of influencing osteochondral defect healing. Specifically, scaffold material elastic modulus values in the range of cancellous bone (low GPa range) and a scaffold architecture that provided stability, i.e., resistance against displacement, in both the main loading direction and perpendicular to it supported the repair process. The here presented model, despite its simplifications, is regarded as a powerful tool to screen for promising properties of novel scaffold candidates fostering osteochondral defect regeneration prior to their implementation in vivo

Design und Charakterisierung von Multiskalen-Hybridscaffolds für die enchondrale Ossifikation

Critical size defects in bone and osteochondral defects in articular cartilage do not heal without clinical intervention. Current clinical treatments of both defect types are associated with strong limitations, which could be addressed by the development of tissue engineering (TE) treatment strategies. Collagen scaffolds with a highly aligned architecture have been previously shown to induce bone formation by endochondral ossification in large bone defects in vivo. The establishment of the endochondral ossification process has been proven to depend on the aligned architecture of the collagen scaffolds, without the need for the inclusion of additional biochemical factors. However, the direct clinical application of these collagen scaffolds is hindered by their extremely low stiffness (low kPa range), which determines the easy alteration of the aligned architecture by means of tissue forces and extracellular matrix deposition within the scaffold pores. Here, the limitations of the collagen scaffolds with highly aligned architecture are addressed by incorporation of a stiffer synthetic support structure, generating multiscale hybrid scaffolds. The aim of the support structure is not limited to the improvement of the mechanical stiffness of the scaffold system at tissue level, but it extends also to the steering of the tissue regeneration process by means of different scaffold-dependent mechanical cues, which could be achieved by different types of support structures, e.g. a stiff and a compliant one. In fact, the development of bone through endochondral ossification happens by first establishing a cartilaginous template, which is then mineralized. Moreover, the formation of bone and cartilage has been associated to mechanical stimuli of lower and higher magnitude, respectively. Therefore, mechanical cues determined by the stiffness at tissue level of the hybrid scaffolds are intended to be used to guide tissue formation towards either bone or cartilage. The successful establishment of this approach would enable the use of controlled mechanics for applications beyond bone defect healing, e.g. in the treatment of osteochondral defects. In this thesis, a stiff and a compliant support structure to be included in the hybrid scaffolds were designed. Thereafter, the production of the support structures by selective laser sintering from poly(ε-caprolactone) was optimized in terms of material choice and resulting support structure properties. Moreover, stiff and compliant support structure architectures with significant differences in stiffness and fatigue resistance in in vivo-like conditions were designed. Subsequently, stiff and compliant hybrid scaffolds were produced and characterized in terms of morphology of the collagen walls, mechanical properties, and in vitro cell-material interactions. Concurrently, the ideal mechanical and architectural properties of scaffolds for osteochondral defect regeneration were here investigated by means of a computational model.Die Heilung von großen Knochendefekten, sowie osteochondralen Defekten ist gegenwärtig stets auf eine klinische Intervention angewiesen. Allerdings weisen derzeitige Behandlungsmethoden von beiden Defekten erhebliche Limitationen auf. Die Entwicklung von neue Methoden basierend auf „Tissue Engineering“ (TE) könnten dazu beitragen, diese Limitationen auszugleichen. Für kollagen-basierte Biomaterialien mit einer gerichteten Porenstruktur wurde kürzlich gezeigt, dass sie eine endochondrale Ossifikation anregen können. Die Implantation dieses Biomaterials in einen großen Knochendefekt im Femur der Ratte führte zu einer Knochenbildung, die rein auf die Architektur des Materials zurückzuführen ist und ohne die zusätzliche Behandlung mit biochemischen Faktoren auskommt. Der Nachteil dieser Biomaterialien ist allerdings ihre extrem geringe mechanische Stabilität resultierend aus einer niedrigen Steifigkeit. Diese Eigenschaft macht die direkte Anwendung dieser Scaffolds in großen Knochendefekten zunächst problematisch, da die gerichtete Porenstruktur durch die im Gewebe vorhandenen mechanischen Kräfte verloren gehen kann. Ziel dieser Arbeit war es, diese Limitation des Kollagenscaffolds durch eine zusätzliche Inkorporation einer synthetischen Stützstruktur mit höherer Steifigkeit zu überwinden und dadurch einen Hybridscaffold mit multiskalaren Eigenschaften zu entwickeln. Die Stützstruktur sollte hierbei nicht nur allein die mechanische Stabilität des Scaffolds verbessern, sondern auch je nach mechanischem Stimuli entweder den Heilungsprozess nach Knochen- oder Knorpelbildung durch endochondrale Ossifikation abstoppen. Dies hat den Hintergrund, dass die Gewebeeigenschaften in Entwicklung von Knochen und Knorpelgewebe in vivo mit unterschiedlichen mechanischen Eigenschaften assoziiert sind. In diesem Projekt wurden die Zielgewebe-spezifischen mechanischen Stimuli durch unterschiedliche Designs, und damit auch unterschiedlichen Steifigkeiten (z.B. steif und weich), der Stützstrukturen erreicht. Wäre diese Methode erfolgreich, könnten die Hybdriscaffolds als Behandlung von Knochendefekten kritischer Größe, sowie osteochondraler Defekte benutzt werden. In dieser Arbeit wurden je eine weiche und eine steife Stützstruktur für einen Hybridscaffold, die verschiedene Steifigkeit hatten und die in vivo Lasten tragen könnten, entwickelt. Danach waren steife und weiche Hybdridscaffolds hergestellt und ihren Eigenschaften (Kollagen Struktur, Steifigkeit, in vitro Zell-Materialen Interaktionen) gemessen worden. Außerdem wurde ein Computermodel etabliert, um die idealen mechanischen und strukturellen Eigenschaften eines Scaffolds für osteochondrale Defekte zu erforschen

Inner strut morphology is the key parameter in producing highly porous and mechanically stable poly(ε-caprolactone) scaffolds via selective laser sintering

Selective laser sintering (SLS) is an established method to produce dimensionally accurate scaffolds for tissue engineering (TE) applications, especially in bone. In this context, the FDA-approved, biodegradable polymer poly (ε-caprolactone) (PCL) has been suggested as a suitable scaffold material. However, PCL scaffold mechanical stability – an attribute of particular importance in the field of bone TE – was not considered as a primary target for SLS process parameters optimization so far. Here, we investigated the influence of SLS process parameters on the sintered scaffolds with the aim of producing highly porous (>70% porosity) PCL scaffolds with sub-mm geometrical features for bone TE. Specifically, we studied the influence of laser power, beam compensation and laser beam diameter on the dimensional accuracy and mechanical stiffness of the produced PCL scaffolds. We found that the ratio between the diameter of the molten cross-section within scaffold struts and the outer strut diameter (including partially sintered particles) depended on the SLS process parameters. By maximizing this ratio, the mechanical stability could be optimized. The comparison with in silico predictions of scaffold me-chanical stiffness revealed that the diameter of the molten cross-section within struts and not the strut diameter controlled the mechanical behaviour of the scaffold. These observations should be considered when evaluating the quality of the sintering process based on dimensional accuracy, especially for features <1 mm. Based on these findings, we suggested an approach to evaluate the sintering outcome and to define SLS process parameters that enable the production of highly porous scaffolds that are both dimensionally accurate and mechanically stable. Moreover, the cytocompatibility of PCL scaffolds was evaluated by elution tests with primary human mesen-chymal stromal cells. No evidence of cytotoxicity was found in any of the investigated scaffolds, confirming the suitability of SLS as production technique of PCL scaffolds for bone TE over a wide range of SLS process parameters

Custom-made poly(urethane) coatings improve the mechanical properties of bioactive glass scaffolds designed for bone tissue engineering

The replication method is a widely used technique to produce bioactive glass (BG) scaffolds mimicking trabecular bone. However, these scaffolds usually exhibit poor mechanical reliability and fast degradation, which can be improved by coating them with a polymer. In this work, we proposed the use of custom-made poly(urethane)s (PURs) as coating materials for 45S5 Bioglass®-based scaffolds. In detail, BG scaffolds were dip-coated with two PURs differing in their soft segment (poly(ε-caprolactone) or poly(ε-caprolactone)/poly(ethylene glycol) 70/30 w/w) (PCL-PUR and PCL/PEG-PUR) or PCL (control). PUR-coated scaffolds exhibited biocompatibility, high porosity (ca. 91%), and improved mechanical properties compared to BG scaffolds (2–3 fold higher compressive strength). Interestingly, in the case of PCL-PUR, compressive strength significantly increased by coating BG scaffolds with an amount of polymer approx. 40% lower compared to PCL/PEG-PUR- and PCL-coated scaffolds. On the other hand, PEG presence within PCL/PEG-PUR resulted in a fast decrease in mechanical reliability in an aqueous environment. PURs represent promising coating materials for BG scaffolds, with the additional pros of being ad-hoc customized in their physico-chemical properties. Moreover, PUR-based coatings exhibited high adherence to the BG surface, probably because of the formation of hydrogen bonds between PUR N-H groups and BG surface functionalities, which were not formed when PCL was used

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Efficient therapeutic options are needed to control the spread of SARS-CoV-2 that has caused more than 922,000 fatalities as of September 13th, 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block ACE2 attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Cocktails including S2M11, S2E12 or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.elocation-id: eabe3354status: Published onlin

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49&nbsp;days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics