Towards osteochondral regeneration with human bone marrow derived mesenchymal stromal cells in a functionalized hydrogel system

There is the need of alternative treatment strategies for osteochondral injuries that include a defect of articular cartilage and the underlying bone. Human bone marrow derived mesenchymal stromal cells (BMSCs) due to their ease of isolation and multipotent differentiation capacity have been investigated for a long time as cell source candidate for osteochondral tissue engineering. However, their clinical application has been hampered by several limitations most importantly such as intrinsic tendency to acquire a (pre-) hypertrophic chondrogenic phenotype leading to endochondral ossification in vivo, lack of spatial control of the differentiated cell phenotypes and vast donor-to-donor variability, as well as unpredictability of differentiation outcome potentially due to the crude isolation procedure and lack of selective markers. Part I of the thesis addressed the optimization of the protocol to generate endochondral bone by BMSCs and the assessment of the formation of bone-cartilage composites by combination of BMSCs with nasal chondrocytes (NCs). To this end, an enzymatically cross-linked and cell-degradable poly(ethylene glycol) (PEG) based hydrogel system served as a scaffolding material. By functionalization of the hydrogel with TGFß3 employing an affinity binding strategy, encapsulated BMSCs were induced to undergo endochondral ossification resulting in the efficient formation of ossicles including a cortical rim and bone marrow upon immediate subcutaneous implantation in immunocompromised mice. This demonstrated that the otherwise needed lengthy in vitro culture step can be circumvented. In bi-layered hydrogels endochondral ossification of BMSCs occurred similarly to the single-layered configuration, while NCs formed cartilaginous tissue, however, unexpectedly acquired hypertrophic features under the influence of the TGFß3 from the BMSC-layer. Replacing TGFß3 with BMP-2 allowed the formation of an osteochondral construct including hyaline cartilage corroborating the potential of our approach to generate cartilage-bone composites. In future, these bi-layered gels need to be tested in an orthotopic model with special focus on how an interface closely resembling the native one can be generated. Part II of the thesis aimed at elucidating the existence of an expanded BMSC subpopulation with superior chondrogenic differentiation potential. It was hypothesized that retrospective analysis of single clones with high chondrogenic capacity have a different gene expression profile than clones with low capacity and that differential gene expression would guide to prospectively isolate superior chondrogenic potential clones from bulk BMSCs. For one of the tested donors a segregation of clones of high and low CC based on their transcriptomic profile could be observed. Comparison of sorted multiclonal BMSCs based on CD56/NCAM1 - the most promising surface marker identified by the transcriptomic analysis - in chondrogenic in vitro culture assays showed a trend of better chondrogenesis in the CD56+ cells, however, it necessitates confirmation with additional donors. In a further analysis of clones from other donors, intra-donor variability compromised the revelation of transcriptional signatures of clones with high versus low chondrogenic capacity. In future, RNA sequencing as well as cell sorting are required to be performed at earlier time points to exclude confounding effects from extensive cell expansion. Ultimately, identification of a cell subset with superior chondrogenic potential may aid to develop improved BMSC based osteochondral tissue engineering approaches

SARS-CoV-2 infects epithelial cells of the blood-cerebrospinal fluid barrier rather than endothelial cells or pericytes of the blood-brain barrier.

BACKGROUND As a consequence of SARS-CoV-2 infection various neurocognitive and neuropsychiatric symptoms can appear, which may persist for several months post infection. However, cell type-specific routes of brain infection and underlying mechanisms resulting in neuroglial dysfunction are not well understood. METHODS Here, we investigated the susceptibility of cells constituting the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP) to SARS-CoV-2 infection using human induced pluripotent stem cell (hiPSC)-derived cellular models and a ChP papilloma-derived epithelial cell line as well as ChP tissue from COVID-19 patients, respectively. RESULTS We noted a differential infectibility of hiPSC-derived brain microvascular endothelial cells (BMECs) depending on the differentiation method. Extended endothelial culture method (EECM)-BMECs characterized by a complete set of endothelial markers, good barrier properties and a mature immune phenotype were refractory to SARS-CoV-2 infection and did not exhibit an activated phenotype after prolonged SARS-CoV-2 inoculation. In contrast, defined medium method (DMM)-BMECs, characterized by a mixed endothelial and epithelial phenotype and excellent barrier properties were productively infected by SARS-CoV-2 in an ACE2-dependent manner. hiPSC-derived brain pericyte-like cells (BPLCs) lacking ACE2 expression were not susceptible to SARS-CoV-2 infection. Furthermore, the human choroid plexus papilloma-derived epithelial cell line HIBCPP, modeling the BCSFB was productively infected by SARS-CoV-2 preferentially from the basolateral side, facing the blood compartment. Assessment of ChP tissue from COVID-19 patients by RNA in situ hybridization revealed SARS-CoV-2 transcripts in ChP epithelial and ChP stromal cells. CONCLUSIONS Our study shows that the BCSFB of the ChP rather than the BBB is susceptible to direct SARS-CoV-2 infection. Thus, neuropsychiatric symptoms because of COVID-19 may rather be associated with dysfunction of the BCSFB than the BBB. Future studies should consider a role of the ChP in underlying neuropsychiatric symptoms following SARS-CoV-2 infection

Learn, simplify and implement: developmental re-engineering strategies for cartilage repair

The limited self-healing capacity of cartilage in adult individuals, and its tendency to deteriorate once structurally damaged, makes the search for therapeutic strategies following cartilage-related traumas relevant and urgent. To date, autologous cell-based therapies represent the most advanced treatments, but their clinical success is still hampered by the long-term tendency to form fibrous as opposed to hyaline cartilage tissue. Would the efficiency and robustness of therapies be enhanced if cartilage regeneration approaches were based on the attempt to recapitulate processes occurring during cartilage development ("developmental engineering")? And from this perspective, shouldn't cartilage repair strategies be inspired by development, but adapted to be effective in a context (an injured joint in an adult individual) that is different from the embryo ("developmental re-engineering")? Here, starting from mesenchymal stem/stromal cells (MSCs) as an adult cell source possibly resembling features of the embryonic mesenchyme, we propose a developmental re-engineering roadmap based on the following three steps: (i) learn from embryonic cartilage development which are the key pathways involved in MSC differentiation towards stable cartilage, (ii) simplify the complex developmental events by approximation to essential molecular pathways, possibly by using in vitro high-throughput models and, finally, (iii) implement the outcomes at the site of the injury by establishing an appropriate interface between the delivered signals and the recipient environment (e.g., by controlling inflammation and angiogenesis). The proposed re-design of developmental machinery by establishing artificial developmental events may offer a chance for regeneration to those tissues, like cartilage, with limited capacity to recover from injuries

COVID-19 and the Vasculature: Current Aspects and Long-Term Consequences

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was first identified in December 2019 as a novel respiratory pathogen and is the causative agent of Corona Virus disease 2019 (COVID-19). Early on during this pandemic, it became apparent that SARS-CoV-2 was not only restricted to infecting the respiratory tract, but the virus was also found in other tissues, including the vasculature. Individuals with underlying pre-existing co-morbidities like diabetes and hypertension have been more prone to develop severe illness and fatal outcomes during COVID-19. In addition, critical clinical observations made in COVID-19 patients include hypercoagulation, cardiomyopathy, heart arrythmia, and endothelial dysfunction, which are indicative for an involvement of the vasculature in COVID-19 pathology. Hence, this review summarizes the impact of SARS-CoV-2 infection on the vasculature and details how the virus promotes (chronic) vascular inflammation. We provide a general overview of SARS-CoV-2, its entry determinant Angiotensin-Converting Enzyme II (ACE2) and the detection of the SARS-CoV-2 in extrapulmonary tissue. Further, we describe the relation between COVID-19 and cardiovascular diseases (CVD) and their impact on the heart and vasculature. Clinical findings on endothelial changes during COVID-19 are reviewed in detail and recent evidence from in vitro studies on the susceptibility of endothelial cells to SARS-CoV-2 infection is discussed. We conclude with current notions on the contribution of cardiovascular events to long term consequences of COVID-19, also known as “Long-COVID-syndrome”. Altogether, our review provides a detailed overview of the current perspectives of COVID-19 and its influence on the vasculature

Spatially confined induction of endochondral ossification by functionalized hydrogels for ectopic engineering of osteochondral tissues

Despite the various reported approaches to generate osteochondral composites by combination of different cell types and materials, engineering of templates with the capacity to autonomously and orderly develop into cartilage-bone bi-layered structures remains an open challenge. Here, we hypothesized that the embedding of cells inducible to endochondral ossification (i.e. bone marrow derived mesenchymal stromal cells, BMSCs) and of cells capable of robust and stable chondrogenesis (i.e. nasal chondrocytes, NCs) adjacent to each other in bi-layered hydrogels would develop directly in vivo into osteochondral tissues. Poly(ethylene glycol) (PEG) hydrogels were functionalized with TGFβ3 or BMP-2, enzymatically polymerized encapsulating human BMSCs, combined with a hydrogel layer containing human NCs and ectopically implanted in nude mice without pre-culture. The BMSC-loaded layers reproducibly underwent endochondral ossification and generated ossicles containing bone and marrow. The NC-loaded layers formed cartilage tissues, which (under the influence of BMP-2 but not of TGFβ3 from the neighbouring layer) remained phenotypically stable. The proposed strategy, resulting in orderly connected osteochondral composites, should be further assessed for the repair of osteoarticular defects and will be useful to model developmental processes leading to cartilage-bone interfaces