The symbiotic effect of osteoinductive extracellular vesicles and mineralized microenvironment on osteogenesis.

The increasing prevalence of bone-related diseases has raised concern about the need for an osteoinductive and mechanically stronger scaffold-based bone tissue engineering (BTE) alternative. A mineralized microenvironment, similar to the native bone microenvironment, is required in the scaffold to recruit and differentiate local mesenchymal stem cells at the bone defect site. Further, extracellular vesicles (EVs), pre-osteoblasts' secretome, contain osteoinductive cargo and have recently been exploited in bone regeneration. This work developed a cell-free and mechanically strong interpenetrating network-based scaffold for BTE by combining the action of osteoinductive EVs with a mineralized microenvironment. The MC3T3 (a pre-osteoblast cell line) is used as a source of EVs and as the target population. The optimal concentration of MC3T3-EVs was first determined to induce osteogenesis in target cells. The osteoinductive potential of the scaffold was estimated in vitro by osteogenesis-related markers like the alkaline phosphatase (ALP) enzyme and calcium content. The MC3T3-EVs cargo was also studied for osteoinductive signals such as ALP, calcium, and mRNA. The findings of this work indicated that MC3T3-EVs at a 90 µg/mL dose had significantly higher ALP activity than 0 µg/mL (1.47-fold), 10 µg/mL (1.41-fold), and 30 µg/mL (1.39-fold) EV-concentration on day 14. Further combination of the optimum dose of EVs with a mineralized microenvironment significantly enhanced ALP activity (1.5-fold) and mineralization (3.36-fold) as compared to the control group on day 7. EV cargo analysis revealed the presence of calcium, the ALP enzyme, and the mRNAs necessary for osteogenesis and angiogenesis. ALP activity was significantly boosted in the EV-containing target cells as early as day 1, and mineralization began on day 7 because MC3T3-EVs carry ALP enzymes and calcium as cargo. When osteoinductive EVs were combined with an osteoconductive mineralized microenvironment, osteogenesis was significantly enhanced in target cells at early time points. The interaction between osteoinductive EVs and the mineralized milieu facilitates the process of osteogenesis in the target cells and suggests a potential cell-free strategy for in vivo bone repair.

Cell-Instructive Mineralized Microenvironment Regulates Osteogenesis: A Growing SYMBIOSIS of Cell Biology and Biomaterials Engineering in Bone Tissue Regeneration.

One of the objectives of bone tissue engineering is to produce scaffolds that are biocompatible, osteoinductive, and mechanically equivalent to the natural extracellular matrix of bone in terms of structure and function. Reconstructing the osteoconductive bone microenvironment into a scaffold can attract native mesenchymal stem cells and differentiate them into osteoblasts at the defect site. The symbiotic relationship between cell biology and biomaterial engineering could result in composite polymers containing the necessary signals to recreate tissue- and organ-specific differentiation. In the current work, drawing inspiration from the natural stem cell niche to govern stem cell fate, the cell-instructive hydrogel platforms were constructed by engineering the mineralized microenvironment. This work employed two different hydroxyapatite delivery strategies to create a mineralized microenvironment in an alginate-PEGDA interpenetrating network (IPN) hydrogel. The first approach involved coating of nano-hydroxyapatite (nHAp) on poly(lactide-co-glycolide) microspheres and then encapsulating the coated microspheres in an IPN hydrogel for a sustained release of nHAp, whereas the second approach involved directly loading nHAp into the IPN hydrogel. This study demonstrate that both direct encapsulation and a sustained release approach showed enhanced osteogenesis in target-encapsulated cells; however, direct loading of nHAp into the IPN hydrogel increased the mechanical strength and swelling ratio of the scaffold by 4.6-fold and 1.14-fold, respectively. In addition, the biochemical and molecular studies revealed improved osteoinductive and osteoconductive potential of encapsulated target cells. Being less expensive and simple to perform, this approach could be beneficial in clinical settings.

Dexamethasone release pattern via a three-dimensional system for effective bone regeneration.

For over a decade, dexamethasone (DEX) has been used for bone regenerative and anti-inflammatory purposes. It has also shown promise for inducing bone regeneration by using it as component of osteoinductive differentiation medium, particularly forin vitroculture models. Despite its osteoinductive properties, its use is limited due to its associated cytotoxicity, particularly when used at higher concentrations. DEX has adverse effects when taken orally; thus, it is best to use it in a targeted manner. Even when given locally, the pharmaceutical should be distributed in a controlled manner based on the needs of the wounded tissue. However, because drug activity is assessed in two-dimensional (2D) circumstances and the target tissue is a three-dimensional (3D) structure, assessing DEX activity and dosage in a 3D milieu is critical for bone tissue development. The current review examines the advantages of a 3D approach over traditional 2D culture methods and delivery devices for controlled DEX delivery, particularly for bone repair. Further, this review explores the latest advancement and challenges in biomaterial-based therapeutic delivery approaches for bone regeneration. This review also discusses possible future biomaterial-based strategies to study efficient DEX delivery.

Well-orchestrated physico-chemical and biological factors for enhanced secretion of osteogenic and angiogenic extracellular vesicles by mesenchymal stem cells in a 3D culture format.

The secretome of mesenchymal stem cells (MSCs) is being studied for its regenerative potential for the treatment of various disorders, including bone diseases. However, mimicking the physiological parameters of native bone could further improve MSCs' secretory profile. The proteomic analysis revealed that MSCs have a diverse secretory profile depending on the cell formats used to grow them, such as two-dimensional (2D) or three-dimensional (3D) microenvironments. Stem cells are given biochemical and biophysical stimuli in a 3D milieu that mimics in vivo situations. Compared to the gold standard monolayer culture, extracellular vesicles (EVs) released under 3D conditions improved the EV cargo numerically and qualitatively. The higher requirements of EVs in clinical trials with consistent therapeutic potential are challenging. This review discusses the impact of cell culture formats on the regenerative potential of MSCs, specifically in bone regeneration. The poor yield and heterogeneity issues have hampered the therapeutic usage of EVs. Therefore, this review further explores various engineering approaches that could enhance EVs' scalability from MSCs and their therapeutic effectiveness beyond their native utility in bone tissue regeneration. This review also highlights some of the upcoming 3D approaches/models that might be useful for the enhanced secretion of therapeutic EVs from stem cells. Finally, we discuss possible future directions and conclusions in this domain.

Phylogenetic diversity and activity screening of cultivable Actinobacteria isolated from marine sponges and associated environments from the western coast of India.

The phylogenetic diversity of cultivable actinobacteria isolated from sponges (Haliclona spp.) and associated intertidal zone environments along the northern parts of the western coast of India were studied using 16S rRNA gene sequences. A subset of randomly selected actinobacterial cultures were screened for three activities, namely predatory behaviour, antibacterial activity and enzyme inhibition. We recovered 237 isolates from the phylum Actinobacteria belonging to 19 families and 28 genera, which could be attributed to 95 putative species using maximum-likelihood partition and 100 putative species using Bayesian partition in Poisson tree processes. Although the trends in the discovery of actinobacterial genera isolated from sponges were consistent with previous studies from different study areas, we provide the first report of nine actinobacterial species from sponges. We observed widespread non-obligate epibiotic predatory behaviour in eight actinobacterial genera and we provide the first report of predatory activity in Brevibacterium , Glutamicibacter , Micromonospora , Nocardiopsis , Rhodococcus and Rothia . Sponge-associated actinobacteria showed significantly more predatory behaviour than environmental isolates. While antibacterial activity by actinobacterial isolates mainly affected Gram-positive target bacteria with little or no effect on Gram-negative bacteria, predation targeted both Gram-positive and Gram-negative prey with equal propensity. Actinobacterial isolates from both sponges and associated environments produced inhibitors of serine proteases and angiotensin-converting enzyme. Predatory behaviour was strongly associated with inhibition of trypsin and chymotrypsin. Our study suggests that the sponges and associated environments of the western coast of India are rich in actinobacterial diversity, with widespread predatory activity, antibacterial activity and production of enzyme inhibitors. Understanding the diversity and associations among various actinobacterial activities - with each other and the source of isolation - can provide new insights into marine microbial ecology and provide opportunities to isolate novel therapeutic agents.

Hydrogel-Assisted 3D Model to Investigate the Osteoinductive Potential of MC3T3-Derived Extracellular Vesicles.

Effective and rapid regeneration of bone defects often pose substantial challenges in severe accidental injuries and disabilities occurring due to diseases and/or advanced age, especially in patients having reduced tissue regeneration competence. The success of mesenchymal stromal cell (MSC)-based research strategies in improving bone regeneration was hampered not only due to the limited knowledge of therapeutic actions of MSCs but also due to difficulties as well as expenses related to cell manufacturing and time taken for ethical approvals for clinical use of living cells and engineered tissues. The recent trend indicated that there is a shift from the direct usage of MSCs toward the application of paracrine factors and extracellular vesicles (EVs) isolated from their MSC secretome in bone tissue regeneration. This shift has directed research into the development of "cell-free" therapeutics, which could be a better alternative due to its several advantages over the use of their parental MSCs. Furthermore, accumulating evidence suggested that the 3D microenvironment influences the paracrine effects of MSCs. Although the osteogenic role of EVs has been explored recently, the current study showed, for the first time, that encapsulation of EVs along with MC3T3 cells in a 3D hydrogel-assisted culture with a distinct porous microenvironment having meso and macro (0.05-200 µm) pore size distribution resulted in an improved osteogenic response in vitro. The present work was primarily focused on investigating the influence of EVs isolated under distinct priming conditions to enhance the osteogenic potential. In addition, in the current work, the osteogenic ability of different types of EVs (microvesicles and exosomes) and total EVs isolated at different time points was also examined when encapsulated with MC3T3 cells in an alginate gel. Using various biochemical assays, such as alkaline phosphatase (ALP) production and calcium secretion, it was observed that both microvesicles and exosomes collected from MC3T3 cells independently had osteogenic potential; however, their collective activity was found to be superior. We further showed that EVs induce an early osteogenic response in MC3T3 cells as indicated by ALP and calcium secretion at a much earlier time point, compared to the controls. Our data suggested that this 3D hydrogel-assisted system provides close proximity of cells and EVs, and thus, mimics the in vivo scenario, making it clinically useful for bone tissue engineering.