3D Niche-Inspired Scaffolds as a Stem Cell Delivery System for the Regeneration of the Osteochondral Interface.

The regeneration of the osteochondral unit represents a challenge due to the distinct cartilage and bone phases. Current strategies focus on the development of multiphasic scaffolds that recapitulate features of this complex unit and promote the differentiation of implanted bone-marrow derived stem cells (BMSCs). In doing so, challenges remain from the loss of stemness during in vitro expansion of the cells and the low control over stem cell activity at the interface with scaffolds in vitro and in vivo. Here, this work scaffolds inspired by the bone marrow niche that can recapitulate the natural healing process after injury. The construct comprises an internal depot of quiescent BMSCs, mimicking the bone marrow cavity, and an electrospun (ESP) capsule that "activates" the cells to migrate into an outer "differentiation-inducing" 3D printed unit functionalized with TGF-ß and BMP-2 peptides. In vitro, niche-inspired scaffolds retained a depot of nonproliferative cells capable of migrating and proliferating through the ESP capsule. Invasion of the 3D printed cavity results in location-specific cell differentiation, mineralization, secretion of alkaline phosphatase (ALP) and glycosaminoglycans (GAGs), and genetic upregulation of collagen II and collagen I. In vivo, niche-inspired scaffolds are biocompatible, promoted tissue formation in rat subcutaneous models, and regeneration of the osteochondral unit in rabbit models.

The role of plasma-induced surface chemistry on polycaprolactone nanofibers to direct chondrogenic differentiation of human mesenchymal stem cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are extensively being utilized for cartilage regeneration owing to their excellent differentiation potential and availability. However, controlled differentiation of BMSCs towards cartilaginous phenotypes to heal full-thickness cartilage defects remains challenging. This study investigates how different surface properties induced by either coating deposition or biomolecules immobilization onto nanofibers (NFs) could affect BMSCs chondro-inductive behavior. Accordingly, electrospun poly(ε-caprolactone) (PCL) NFs were exposed to two surface modification strategies based on medium-pressure plasma technology. The first strategy is plasma polymerization, in which cyclopropylamine (CPA) or acrylic acid (AcAc) monomers were plasma polymerized to obtain amine- or carboxylic acid-rich NFs, respectively. The second strategy uses a combination of CPA plasma polymerization and a post-chemical technique to immobilize chondroitin sulfate (CS) onto the NFs. These modifications could affect surface roughness, hydrophilicity, and chemical composition while preserving the NFs' nano-morphology. The results of long-term BMSCs culture in both basic and chondrogenic media proved that the surface modifications modulated BMSCs chondrogenic differentiation. Indeed, the incorporation of polar groups by different modification strategies had a positive impact on the cell proliferation rate, production of the glycosaminoglycan matrix, and expression of extracellular matrix proteins (collagen I and collagen II). The chondro-inductive behavior of the samples was highly dependent on the nature of the introduced polar functional groups. Among all samples, carboxylic acid-rich NFs promoted chondrogenesis by higher expression of aggrecan, Sox9, and collagen II with downregulation of hypertrophic markers. Hence, this approach showed an intrinsic potential to have a non-hypertrophic chondrogenic cell phenotype.

Corrigendum to "A quantitative method to analyse F-actin distribution in cells," [MethodsX 6 (2019), 2562-2569].

[This corrects the article DOI: 10.1016/j.mex.2019.10.018.].

Additive Manufacturing of α-Amino Acid Based Poly(ester amide)s for Biomedical Applications.

α-Amino acid based polyester amides (PEAs) are promising candidates for additive manufacturing (AM), as they unite the flexibility and degradability of polyesters and good thermomechanical properties of polyamides in one structure. Introducing α-amino acids in the PEA structure brings additional advantages such as (i) good cytocompatibility and biodegradability, (ii) providing strong amide bonds, enhancing the hydrogen-bonding network, (iii) the introduction of pendant reactive functional groups, and (iv) providing good cell-polymer interactions. However, the application of α-amino acid based PEAs for AM via fused deposition modeling (FDM), an important manufacturing technique with unique processing characteristics and requirements, is still lacking. With the aim to exploit the combination of these advantages in the creation, design, and function of additively manufactured scaffolds using FDM, we report the structure-function relationship of a series of α-amino acid based PEAs. The PEAs with three different molecular weights were synthesized via the active solution polycondensation, and their performance for AM applications was studied in comparison with a commercial biomedical grade copolymer of l-lactide and glycolide (PLGA). The PEAs, in addition to good thermal stability, showed semicrystalline behavior with proper mechanical properties, which were different depending on their molecular weight and crystallinity. They showed more ductility due to their lower glass transition temperature (Tg; 18-20 °C) compared with PLGA (57 °C). The rheology studies revealed that the end-capping of PEAs is of high importance for preventing cross-linking and further polymerization during the melt extrusion and for the steadiness and reproducibility of FDM. Furthermore, our data regarding the steady 3D printing performance, good polymer-cell interactions, and low cytotoxicity suggest that α-amino acid based PEAs can be introduced as favorable polymers for future AM applications in tissue engineering. In addition, their ability for formation of bonelike apatite in the simulated body fluid (SBF) indicates their potential for bone tissue engineering applications.

Steering cell behavior through mechanobiology in 3D: A regenerative medicine perspective.

Mechanobiology, translating mechanical signals into biological ones, greatly affects cellular behavior. Steering cellular behavior for cell-based regenerative medicine approaches requires a thorough understanding of the orchestrating molecular mechanisms, among which mechanotransducive ones are being more and more elucidated. Because of their wide use and highly mechanotransduction dependent differentiation, this review focuses on mesenchymal stromal cells (MSCs), while also briefly relating the discussed results to other cell types. While the mechanotransduction pathways are relatively well-studied in 2D, much remains unknown of the role and regulation of these pathways in 3D. Ultimately, cells need to be cultured in a 3D environment to create functional de novo tissue. In this review, we explore the literature on the roles of different material properties on cellular behavior and mechanobiology in 2D and 3D. For example, while stiffness plays a dominant role in 2D MSCs differentiation, it seems to be of subordinate importance in 3D MSCs differentiation, where matrix remodeling seems to be key. Also, the role and regulation of some of the main mechanotransduction players are discussed, focusing on MSCs. We have only just begun to fundamentally understand MSCs and other stem cells behavior in 3D and more fundamental research is required to advance biomaterials able to replicate the stem cell niche and control cell activity. This better understanding will contribute to smarter tissue engineering scaffold design and the advancement of regenerative medicine.

Actomyosin and the MRTF-SRF pathway downregulate FGFR1 in mesenchymal stromal cells.

Both biological and mechanical signals are known to influence cell proliferation. However, biological signals are mostly studied in two-dimensions (2D) and the interplay between these different pathways is largely unstudied. Here, we investigated the influence of the cell culture environment on the response to bFGF, a widely studied and important proliferation growth factor. We observed that human mesenchymal stromal cells (hMSCs), but not fibroblasts, lose the ability to respond to soluble or covalently bound bFGF when cultured on microfibrillar substrates. This behavior correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates. Inhibition of actomyosin or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 cells. To our knowledge, this is the first time FGFR1 expression is shown to be regulated through a mechanosensitive pathway in hMSCs. These results add to the sparse literature on FGFR1 regulation and potentially aid designing tissue engineering constructs that better control cell proliferation.

Mechanosensitive regulation of stanniocalcin-1 by zyxin and actin-myosin in human mesenchymal stromal cells.

Stanniocalcin-1 (STC1) secreted by mesenchymal stromal cells (MSCs) has anti-inflammatory functions, reduces apoptosis, and aids in angiogenesis, both in vitro and in vivo. However, little is known about the molecular mechanisms of its regulation. Here, we show that STC1 secretion is increased only under specific cell-stress conditions. We find that this is due to a change in actin stress fibers and actin-myosin tension. Abolishment of stress fibers by blebbistatin and knockdown of the focal adhesion protein zyxin leads to an increase in STC1 secretion. To also study this connection in 3D, where few focal adhesions and actin stress fibers are present, STC1 expression was analyzed in 3D alginate hydrogels and 3D electrospun scaffolds. Indeed, STC1 secretion was increased in these low cellular tension 3D environments. Together, our data show that STC1 does not directly respond to cell stress, but that it is regulated through mechanotransduction. This research takes a step forward in the fundamental understanding of STC1 regulation and can have implications for cell-based regenerative medicine, where cell survival, anti-inflammatory factors, and angiogenesis are critical.

Fiber diameter, porosity and functional group gradients in electrospun scaffolds.

Developing, homeostatic, and regenerating tissues are full of various gradients, including mechanical, chemical, porosity and growth-factor gradients. However, it remains challenging to replicate these gradients using common tissue engineering approaches. Here, we use electrospinning to create scaffolds with in-depth gradients. We created a fiber diameter gradient and pore size gradient throughout the depth of electrospun (ESP) scaffolds by a continuous gradient of polymer concentration. As an alternative to this established method, we developed a novel method to create fiber diameter gradients by changing the voltage on both needle and collector, keeping the total voltage constant. In this way, fiber diameter could be changed in a gradient matter by focusing the electrospinning spot. Using this method, we created a fiber diameter and pore size gradient, while keeping all other parameters constant. Lastly, we developed a novel method to create functional group gradients, which can potentially be used in a wide variety of polymer solutions to couple peptides and proteins to ESP scaffolds. A scaffold with an in-depth gradient of functional groups was created by adding functionalized poly(ethylene glycol) additives to the polymer solution, a novel method with potentially wide applications. The techniques demonstrated here could be applied to a wide variety of polymers and applications and can aid in developing physiologically relevant gradient scaffolds.

Dimensionality changes actin network through lamin A/C and zyxin.

Mechanosensing proteins have mainly been investigated in 2D culture platforms, while understanding their regulation in 3D enviroments is critical for tissue engineering. Among mechanosensing proteins, the actin cytoskeleton plays a key role in human mesenchymal stromal cells (hMSCs) activity, but its regulation in 3D tissue engineered scaffolds remains poorly studied. Here, we show that human mesenchymal stromal cells (hMSCs) cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun and additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization and myosin light chain phosphorylation. Together, this strongly suggests that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Lineage commitment was not affected by the lack of these important osteogenic proteins in 3D, as all cells committed to osteogenesis in bi-potential medium. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, and highlights the difference in the regulation of lineage commitment in 3D enviroments. Together, these results can have important implications for future scaffold design for both stiff- and soft tissue engineering constructs.

A quantitative method to analyse F-actin distribution in cells.

Changes in actin structure and distribution are involved in many cellular processes, such as differentiation, proliferation and migration. Differences in cell shape and size make the analysis of actin distribution difficult. Here, we have developed a Fiji macro that analyzes the distribution of actin within the cell, regardless of cell size or shape. The staining intensity is measured along an automatically drawn line over the cell. The intensity data is divided in equal bins, making the analysis insensitive to changes in cell size or shape. We have also created an R script that further processes the acquired data. Together, final data can be acquired within minutes from a set of images, with freely available software. We demonstrate our method with F-actin staining of cytochalasin D treated cells. The advantages of our methods are: •The analysis is not influenced by cell shape or size•All steps in the analysis are shown, and can therefore easily be verified for each image•All software required for the analysis is freely available.

Immunodeficiency in Bloom's Syndrome.

Bloom's syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20-80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR.