The synovial environment steers cartilage deterioration and regeneration.

Osteoarthritis (OA) was recently defined as an epidemic, and the lack of effective treatment is highly correlated to the limited knowledge regarding the underlying pathophysiology. Failure to regenerate upon trauma is thought to be one of the underlying causes for degenerative diseases, including OA. To investigate why lesions within an OA environment fail to heal, a heterogeneous cell population was isolated from the synovial fluid (SF) of OA patients. The cells' ability to undergo processes required for functional tissue regeneration was evaluated in the presence or absence of autologous SF. The obtained mechanistic findings were then used for the development of an immunomodulatory cell treatment, aimed to restore the pro-regenerative environment. Intra-articular injection in a clinical compassionate use study showed that the treatment restored the articular cartilage and joint homeostasis of OA patients. These findings confirm the role of pro-regenerative immune cells and their targeted influence on progenitor cells for degenerative joint disease therapies.

Syndecan-4 and stromal cell-derived factor-1 alpha functionalized endovascular scaffold facilitates adhesion, spreading and differentiation of endothelial colony forming cells and functions under flow and shear stress conditions.

Acellular vascular scaffolds with capture molecules have shown great promise in recruiting circulating endothelial colony forming cells (ECFCs) to promote in vivo endothelialization. A microenvironment conducive to cell spreading and differentiation following initial cell capture are key to the eventual formation of a functional endothelium. In this study, syndecan-4 and stromal cell-derived factor-1 alpha were used to functionalize an elastomeric biomaterial composed of poly(glycerol sebacate), Silk Fibroin and Type I Collagen, termed PFC, to enhance ECFC-material interaction. Functionalized PFC (fPFC) showed significantly greater ECFCs capture capability under physiological flow. Individual cell spreading area on fPFC (1474 ± 63 µm2 ) was significantly greater than on PFC (1187 ± 54 µm2 ) as early as 2 h, indicating enhanced cell-material interaction. Moreover, fPFC significantly upregulated the expression of endothelial cell specific markers such as platelet endothelial cell adhesion molecule (24-fold) and Von Willebrand Factor (11-fold) compared with tissue culture plastic after 7 days, demonstrating differentiation of ECFCs into endothelial cells. fPFC fabricated as small diameter conduits and tested using a pulsatile blood flow bioreactor were stable and maintained function. The findings suggest that the new surface functionalization strategy proposed here results in an endovascular material with enhanced endothelialization.

Nanocarriers, Progenitor Cells, Combinational Approaches, and New Insights on the Retinal Therapy.

Progenitor cells derived from the retinal pigment epithelium (RPECs) have shown promise as therapeutic approaches to degenerative retinal disorders including diabetic retinopathy, age-related macular degeneration and Stargardt disease. However, the degeneration of Bruch's membrane (BM), the natural substrate for the RPE, has been identified as one of the major limitations for utilizing RPECs. This degeneration leads to decreased support, survival and integration of the transplanted RPECs. It has been proposed that the generation of organized structures of nanofibers, in an attempt to mimic the natural retinal extracellular matrix (ECM) and its unique characteristics, could be utilized to overcome these limitations. Furthermore, nanoparticles could be incorporated to provide a platform for improved drug delivery and sustained release of molecules over several months to years. In addition, the incorporation of tissue-specific genes and stem cells into the nanostructures increased the stability and enhanced transfection efficiency of gene/drug to the posterior segment of the eye. This review discusses available drug delivery systems and combination therapies together with challenges associated with each approach. As the last step, we discuss the application of nanofibrous scaffolds for the implantation of RPE progenitor cells with the aim to enhance cell adhesion and support a functionally polarized RPE monolayer.

Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity.

Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behavior of serum expanded progenitor cells, but the underlying mechanism and how this is reflected in vivo remains unknown. Herein, the cellular and molecular changes associated with a cellular phenotype shift induced by serum-free preconditioning of human periosteum-derived cells were investigated. Following BMP-2 stimulation, preconditioned cells displayed enhanced in vivo bone forming capacity, associated with an adapted cellular metabolism together with an elevated expression of BMPR2. Single-cell RNA sequencing confirmed the activation of pathways and transcriptional regulators involved in bone development and fracture healing, providing support for the augmentation of specified skeletal progenitor cell populations. The reported findings illustrate the importance of appropriate in vitro conditions for the in vivo outcome. In addition, BMPR2 represents a promising biomarker for the enrichment of skeletal progenitor cells for in vivo bone regeneration.

Bone regeneration strategies: Engineered scaffolds, bioactive molecules and stem cells current stage and future perspectives.

Bone fractures are the most common traumatic injuries in humans. The repair of bone fractures is a regenerative process that recapitulates many of the biological events of embryonic skeletal development. Most of the time it leads to successful healing and the recovery of the damaged bone. Unfortunately, about 5-10% of fractures will lead to delayed healing or non-union, more so in the case of co-morbidities such as diabetes. In this article, we review the different strategies to heal bone defects using synthetic bone graft substitutes, biologically active substances and stem cells. The majority of currently available reviews focus on strategies that are still at the early stages of development and use mostly in vitro experiments with cell lines or stem cells. Here, we focus on what is already implemented in the clinics, what is currently in clinical trials, and what has been tested in animal models. Treatment approaches can be classified in three major categories: i) synthetic bone graft substitutes (BGS) whose architecture and surface can be optimized; ii) BGS combined with bioactive molecules such as growth factors, peptides or small molecules targeting bone precursor cells, bone formation and metabolism; iii) cell-based strategies with progenitor cells combined or not with active molecules that can be injected or seeded on BGS for improved delivery. We review the major types of adult stromal cells (bone marrow, adipose and periosteum derived) that have been used and compare their properties. Finally, we discuss the remaining challenges that need to be addressed to significantly improve the healing of bone defects.

* Harnessing the Osteogenicity of In Vitro Stem Cell-Derived Mineralized Extracellular Matrix as 3D Biotemplate to Guide Bone Regeneration.

Advanced biomaterials that are capable of guiding robust bone regeneration are highly demanded for translational therapy of bone defects or bone augmentation in clinics. One of the strategic approaches is to produce tissue engineering (TE) constructs that mediate bone regeneration by recapitulating the natural bone formation or healing process. In this study, we aimed at producing devitalized mineralized carriers with augmented bone forming capacity via a modified culture protocol (i.e., culture conditions with high calcium and/or phosphate concentrations) that first promotes cell growth and, subsequently, mineralized extracellular matrix (ECM) deposition by human periosteum-derived osteoprogenitor cells (hPDCs) on additive manufactured three-dimensional (3D) porous titanium (Ti)-based scaffolds. Qualitative and quantitative analysis was performed to characterize the physicochemical properties of the produced devitalized mineralized carriers, as well as their effects as carriers on in vitro cell growth and osteochondrogenic differentiation of hPDCs under a perfusion bioreactor culture set-up. The results showed that the modified culture protocol was useful to produce devitalized mineralized carriers with different amount, distribution, composition, and morphology of mineralized matrix that resembled hydroxyapatite, and exhibited different Ca2+ release kinetics, distinct human bone morphogenetic protein (hBMP)-2, human vascular endothelial growth factor (hVEGF) proteins, and collagen contents. The produced devitalized mineralized carriers supported 3D growth of hPDCs, with minor osteochondrogenic differentiation effects under the perfusion bioreactor culture condition. Subcutaneous implantation of hPDC-seeded devitalized mineralized carriers in athymic nude rats showed nearly five-fold augmentation in the ectopic bone-forming capacity, with no bone induction obtained for unseeded, devitalized mineralized carriers and plain Ti scaffolds. Implantation of devitalized mineralized carriers in critical-sized calvarial defects resulted in encouraging defect bridging as compared with limited defect bridging by plain Ti scaffolds or in empty defects. This defect bridging was not enhanced by implanting hPDC-seeded devitalized mineralized carriers. In conclusion, the investigated modified culture protocol was useful to produce devitalized mineralized carriers with augmented bone-forming capacity, which potentially could aid bone repair or augmentation in clinics.

Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells.

Clinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect.

A Qualitative Model of the Differentiation Network in Chondrocyte Maturation: A Holistic View of Chondrocyte Hypertrophy.

Differentiation of chondrocytes towards hypertrophy is a natural process whose control is essential in endochondral bone formation. It is additionally thought to play a role in several pathophysiological processes, with osteoarthritis being a prominent example. We perform a dynamic analysis of a qualitative mathematical model of the regulatory network that directs this phenotypic switch to investigate the influence of the individual factors holistically. To estimate the stability of a SOX9 positive state (associated with resting/proliferation chondrocytes) versus a RUNX2 positive one (associated with hypertrophy) we employ two measures. The robustness of the state in canalisation (size of the attractor basin) is assessed by a Monte Carlo analysis and the sensitivity to perturbations is assessed by a perturbational analysis of the attractor. Through qualitative predictions, these measures allow for an in silico screening of the effect of the modelled factors on chondrocyte maintenance and hypertrophy. We show how discrepancies between experimental data and the model's results can be resolved by evaluating the dynamic plausibility of alternative network topologies. The findings are further supported by a literature study of proposed therapeutic targets in the case of osteoarthritis.

Early BMP, Wnt and Ca(2+)/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates.

The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the spatiotemporal activation of signalling pathways has hampered the optimisation of in vivo bone formation and subsequent clinical translation. To gain further knowledge regarding the early molecular events governing bone tissue formation, we combined human periosteum derived progenitor cells with three types of clinically used CaP-scaffolds, to obtain constructs with a distinct range of bone forming capacity in vivo. Protein phosphorylation together with gene expression for key ligands and target genes were investigated 24 hours after cell seeding in vitro, and 3 and 12 days post ectopic implantation in nude mice. A computational modelling approach was used to deduce critical factors for bone formation 8 weeks post implantation. The combined Ca(2+)-mediated activation of BMP-, Wnt- and PKC signalling pathways 3 days post implantation were able to discriminate the bone forming from the non-bone forming constructs. Subsequently, a mathematical model able to predict in vivo bone formation with 96% accuracy was developed. This study illustrates the importance of defining and understanding CaP-activated signalling pathways that are required and sufficient for in vivo bone formation. Furthermore, we demonstrate the reliability of mathematical modelling as a tool to analyse and deduce key factors within an empirical data set and highlight its relevance to the translation of regenerative medicine strategies.

In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

Functionalization of tissue engineering scaffolds with in vitro-generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca(2+)) and phosphate (PO4 (3-)) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions.

Mapping calcium phosphate activated gene networks as a strategy for targeted osteoinduction of human progenitors.

Although calcium phosphate-containing biomaterials are promising scaffolds for bone regenerative strategies, the osteoinductive capacity of such materials is poorly understood. In this study, we investigated whether endogenous mechanisms of in vivo calcium phosphate-driven, ectopic bone formation could be identified and used to induce enhanced differentiation in vitro of the same progenitor population. To accomplish this, human periosteum derived cells (hPDCs) were seeded on hydroxyapatite/collagen scaffolds (calcium phosphate rich matrix or CPRM), or on decalcified scaffolds (calcium phosphate depleted matrix or CPDM), followed by subcutaneous implantation in nude mice to trigger ectopic bone formation. In this system, osteoblast differentiation occurred in CPRM scaffolds, but not in CPDM scaffolds. Gene expression was assessed by human full-genome microarray at 20 h after seeding, and 2, 8 and 18 days after implantation. In both matrices, implantation of the cell constructs triggered a similar gene expression cascade, however, gene expression dynamics progressed faster in CPRM scaffolds than in CPDM scaffolds. The difference in gene expression dynamics was associated with differential activation of hub genes and molecular signaling pathways related to calcium signaling (CREB), inflammation (TNFα, NFkB, and IL6) and bone development (TGFß, ß-catenin, BMP, EGF, and ERK signaling). Starting from this set of pathways, a growth factor cocktail was developed that robustly enhanced osteogenesis in vitro and in vivo. Taken together, our data demonstrate that through the identification and subsequent stimulation of genes, proteins and signaling pathways associated with calcium phosphate mediated osteoinduction, a focused approach to develop targeted differentiation protocols in adult progenitor cells can be achieved.