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The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function. [ABSTRACT FROM AUTHOR]

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation. [ABSTRACT FROM AUTHOR]

Phoenix sylvestris Roxb. (Arecaceae) seeds are used in the treatment of diabetes in the traditional system of medicine. The present study evaluated antihyperglycemic and antioxidant activities as well as the total phenolic and flavonoid content of the methanol extract of P. sylvestris seeds (MEPS). The constituents of the extract were identified by GC–MS analysis. MEPS demonstrated strong antioxidant activity against 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) (IC50 = 162.70 ± 14.99 μg) and nitric oxide (NO) (IC50 = 101.56 ± 9.46 μg/ml) free radicals. It also possesses a substantial amount of phenolics and flavonoids. It significantly (p <.05) reduced blood glucose levels in glucose‐loaded and alloxan‐induced diabetic mice at the doses of 150 and 300 mg/kg b.w., respectively. A total of 46 compounds were detected and identified by gas chromatography–mass spectroscopy (GC–MS) analysis, among which 8‐methylisoquinoline N‐oxide (32.82%) was predominant. The phytochemical study by GC–MS revealed that the MEPS possesses compounds which could be related to its antidiabetic and antioxidant activities. To recapitulate, P. sylvestris seeds can be a very good option for antidiabetic and antioxidant activity though further studies are still recommended to figure out the responsible phytochemicals and establish their exact mechanism of action. [ABSTRACT FROM AUTHOR]

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration. [ABSTRACT FROM AUTHOR]

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrowderived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration. [ABSTRACT FROM AUTHOR]

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-β)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1–3 days) stimulation with TGF-β1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-β1 (10 ng/mL). Like TGF-β1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-β1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-β1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-β1 was the most potent GF for inducing chondrogenesis in BMSCs. [ABSTRACT FROM AUTHOR]

Nicotiana plumbaginifolia Viv. is an annual herb of the family Solanaceae, which grows abundantly in the weedy lands of Bangladesh. This plant possesses analgesic, antibacterial, anti-anxiety and hepatoprotective properties, and produces various phenolic compounds including flavonoids. The present study afforded determination of total phenolic and flavonoid contents, and for the first time, the isolation and characterization of highly oxygenated flavonoids, e.g., 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone (1), 3,3',4',5',5,6,7,8-octamethoxyflavone (2, exoticin), 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3) and (3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (4) from the leaves of N. plumbaginifolia. All these flavonoids are rather rare natural products, and only found in a few genera, e.g., Polygonum and Murraya. The structures of the isolated flavonoids were elucidated by comprehensive spectroscopic analyses, e.g., UV, 1H, 13C NMR, DEPT, HSQC, HMBC and MS. [ABSTRACT FROM AUTHOR]