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.

Injectable MMP-sensitive alginate hydrogels as hMSC delivery systems.

Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful tool for tissue-regeneration therapies. In this context, entrapped cells should be able to escape the matrix becoming more available to actively participate in the healing process. Here, we analyzed the performance of proteolytically degradable alginate hydrogels as vehicles for human mesenchymal stem cells (hMSC) transplantation. Alginate was modified with the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG), which did not promote dendritic cell maturation in vitro, neither free nor conjugated to alginate chains, indicating low immunogenicity. hMSC were entrapped within MMP-sensitive and MMP-insensitive alginate hydrogels, both containing cell-adhesion RGD peptides. Softer (2 wt % alginate) and stiffer (4 wt % alginate) matrices were tested. When embedded in a Matrigel layer, hMSC-laden MMP-sensitive alginate hydrogels promoted more extensive outward cell migration and invasion into the tissue mimic. In vivo, after 4 weeks of subcutaneous implantation in a xenograft mouse model, hMSC-laden MMP-sensitive alginate hydrogels showed higher degradation and host tissue invasion than their MMP-insensitive equivalents. In both cases, softer matrices degraded faster than stiffer ones. The transplanted hMSC were able to produce their own collagenous extracellular matrix, and were located not only inside the hydrogels, but also outside, integrated in the host tissue. In summary, injectable MMP-sensitive alginate hydrogels can act as localized depots of cells and confer protection to transplanted cells while facilitating tissue regeneration.

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.

A comparative study of mesenchymal stem cells cultured as cell-only aggregates and in encapsulated hydrogels.

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs.

Installation of click-type functional groups enable the creation of an additive manufactured construct for the osteochondral interface.

Melt extrusion-based additive manufacturing (AM) is often used to fabricate scaffolds for osteochondral (OC) regeneration. However, there are two shortcomings associated with this scaffold manufacturing technique for engineering of tissue interfaces: (a) most polymers used in the processing are bioinert, and (b) AM scaffolds often contain discrete (material) gradients accompanied with mechanically weak interfaces. The inability to mimic the gradual transition from cartilage to bone in OC tissue leads to poor scaffold performance and even failure. We hypothesized that introducing peptide gradients on the surface could gradually guide human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards on osteogenic phenotype. To work towards this goal, we initially manufactured poly(ϵ-caprolactone)-azide (PCLA) and PCL-maleimide (PCLM) scaffolds. The surface exposed click-type functional groups, with a surface concentration in the 102pmol cm-2regime, were used to introduce bone morphogenic protein-2 or transforming growth factor-beta binding peptide sequences to drive hMSC differentiation towards osteogenic or chondrogenic phenotypes, respectively. After 3 weeks of culture in chondrogenic medium, we observed differentiation towards hypertrophic chondrogenic phenotypes with expression of characteristic markers such as collagen X. In osteogenic medium, we observed the upregulation of mineralization markers. In basic media, the chondro-peptide displayed a minor effect on chondrogenesis, whereas the osteo-peptide did not affect osteogenesis. In a subcutaneous rat model, we observed a minimal foreign body response to the constructs, indicating biocompatibility. As proof-of-concept, we finally used a novel AM technology to showcase its potential to create continuous polymer gradients (PCLA and PCLM) across scaffolds. These scaffolds did not display delamination and were mechanically stronger compared to discrete gradient scaffolds. Due to the versatility of the orthogonal chemistry applied, this approach provides a general strategy for the field; we could anchor other tissue specific cues on the clickable groups, making these gradient scaffolds interesting for multiple interfacial tissue applications.

Mussel-Inspired Catechol Functionalisation as a Strategy to Enhance Biomaterial Adhesion: A Systematic Review.

Biomaterials have long been explored in regenerative medicine strategies for the repair or replacement of damaged organs and tissues, due to their biocompatibility, versatile physicochemical properties and tuneable mechanical cues capable of matching those of native tissues. However, poor adhesion under wet conditions (such as those found in tissues) has thus far limited their wider application. Indeed, despite its favourable physicochemical properties, facile gelation and biocompatibility, gellan gum (GG)-based hydrogels lack the tissue adhesiveness required for effective clinical use. Aiming at assessing whether substitution of GG by dopamine (DA) could be a suitable approach to overcome this problem, database searches were conducted on PubMed® and Embase® up to 2 March 2021, for studies using biomaterials covalently modified with a catechol-containing substituent conferring improved adhesion properties. In this regard, a total of 47 reports (out of 700 manuscripts, ~6.7%) were found to comply with the search/selection criteria, the majority of which (34/47, ~72%) were describing the modification of natural polymers, such as chitosan (11/47, ~23%) and hyaluronic acid (6/47, ~13%); conjugation of dopamine (as catechol "donor") via carbodiimide coupling chemistry was also predominant. Importantly, modification with DA did not impact the biocompatibility and mechanical properties of the biomaterials and resulting hydrogels. Overall, there is ample evidence in the literature that the bioinspired substitution of polymers of natural and synthetic origin by DA or other catechol moieties greatly improves adhesion to biological tissues (and other inorganic surfaces).

Embryo stage of development is not decisive for reproductive outcomes in frozen-thawed embryo transfer cycles.

OBJECTIVE: To evaluate if the outcomes of IVF/ICSI in frozen-thawed embryo transfer and fresh embryo transfer cycles differ in relation to cleavage and blastocyst stages. METHODS: Retrospective cohort study to compare IVF/ICSI outcomes between fresh embryo transfer and frozen-thawed embryo transfer cycles, according to the stage of embryo development. Analysis was carried out on 443 consecutive embryo transfer cycles performed between January 1st and December 31st, 2014. Women aged up to 38 and submitted to embryo transfer cycles with fresh (n = 309) or frozen-thawed (n = 134) embryos at a private center for assistance in human reproduction were considered for analysis. Results in each group were stratified according to the stage of embryo development: cleavage stage and blastocyst stage. Main outcome measures were implantation rate, clinical pregnancy rate, ongoing pregnancy rate and live birth rate per cycle. RESULTS: In the fresh embryo transfer group, for cleavage stage versus blastocyst stage, respectively, implantation rates were 22% and 47% (p = 0.0005); clinical pregnancy rates were 34% and 64% (p = 0.0057); the ongoing pregnancy rates were 30% and 61% (p = 0.0046) and live birth rates were 28% and 55% (p = 0.0148). There were no significant differences in the rates between cleavage and blastocyst stages in the frozen-thawed group, neither between fresh and frozen-thawed cleavage embryo transfers nor between fresh and frozen-thawed blastocyst transfers. CONCLUSION: Our results confirm that blastocyst transfer is better than cleavage stage in fresh embryo transfer cycles. In frozen-thawed cycles, cleavage or blastocyst stages seem to offer similar reproductive outcomes.

Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

In situ-forming hydrogels of pectin, a polysaccharide present in the cell wall of higher plants, were prepared using an internal ionotropic gelation strategy based on calcium carbonate/d-glucono-δ-lactone, and explored for the first time as cell delivery vehicles. Since no ultrapure pectins are commercially available yet, a simple and efficient purification method was established, effectively reducing the levels of proteins, polyphenols and endotoxins of the raw pectin. The purified pectin was then functionalized by carbodiimide chemistry with a cell-adhesive peptide (RGD). Its gelation was analyzed by rheometry and optimized. Human mesenchymal stem cells embedded within unmodified and RGD-pectin hydrogels of different viscoelasticities (1.5 and 2.5 wt%) remained viable and metabolically active for up to 14 days. On unmodified pectin hydrogels, cells remained isolated and round-shaped. In contrast, within RGD-pectin hydrogels they elongated, spread, established cell-to-cell contacts, produced extracellular matrix, and migrated outwards the hydrogels. After 7 days of subcutaneous implantation in mice, acellular pectin hydrogels were considerably degraded, particularly the 1.5 wt% hydrogels. Altogether, these findings show the great potential of pectin-based hydrogels, which combine an interesting set of easily tunable properties, including the in vivo degradation profile, for tissue engineering and regenerative medicine.

Correction: Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

Correction for 'Biofunctionalized pectin hydrogels as 3D cellular microenvironments' by Sara C. Neves et al., J. Mater. Chem. B, 2015, 3, 2096-2108.

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells.

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP-alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4weeks, OGP-alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration.