Rapid quality assessment and traceability of ginger powder from Northeast India and Indian market based on near infrared spectroscopic fingerprinting.

INTRODUCTION: Ginger (Zingiber officinale Rosc.) varies widely due to varying concentrations of phytochemicals and geographical origin. Rapid non-invasive quality and traceability assessment techniques ensure a sustainable value chain. OBJECTIVE: The objective of this study is the development of suitable machine learning models to estimate the concentration of 6-gingerol and check traceability based on the spectral fingerprints of dried ginger samples collected from Northeast India and the Indian market using near-infrared spectrometry. METHODS: Samples from the market and Northeast India underwent High Performance Liquid Chromatographic analysis for 6-gingerol content estimation. Near infrared (NIR) Spectrometer acquired spectral data. Quality prediction utilized partial least square regression (PLSR), while fingerprint-based traceability identification employed principal component analysis and t-distributed stochastic neighbor embedding (t-SNE). Model performance was assessed using RMSE and R2 values across selective wavelengths and spectral fingerprints. RESULTS: The standard normal variate pretreated spectral data over the wavelength region of 1,100-1,250 nm and 1,325-1,550 nm showed the optimal calibration model with root mean square error of calibration and R2 C (coefficient of determination for calibration) values of 0.87 and 0.897 respectively. A lower value (0.24) of root mean square error of prediction and a higher value (0.973) of R2 P (coefficient of determination for prediction) indicated the effectiveness of the developed model. t-SNE performed better clustering of samples based on geographical location, which was independent of gingerol content. CONCLUSION: The developed NIR spectroscopic model for Indian ginger samples predicts the 6-gingerol content and provides geographical traceability-based identification to ensure a sustainable value chain, which can promote efficiency, cost-effectiveness, consumer confidence, sustainable sourcing, traceability, and data-driven decision-making.

Assessing the advantages of 3D bioprinting and 3D spheroids in deciphering the osteoarthritis healing mechanism using human chondrocytes and polarized macrophages.

The molecular niche of an osteoarthritic microenvironment comprises the native chondrocytes, the circulatory immune cells, and their respective inflammatory mediators. Although M2 macrophages infiltrate the joint tissue during osteoarthritis (OA) to initiate cartilage repair, the mechanistic crosstalk that dwells underneath is still unknown. Our study established a co-culture system of human OA chondrocytes and M2 macrophages in 3D spheroids and 3D bioprinted silk-gelatin constructs. It is already well established that Silk fibroin-gelatin bioink supports chondrogenic differentiation due to upregulation of the Wnt/ß-catenin pathway. Additionally, the presence of anti-inflammatory M2 macrophages significantly upregulated the expression of chondrogenic biomarkers (COL-II, ACAN) with an attenuated expression of the chondrocyte hypertrophy (COL-X), chondrocyte dedifferentiation (COL-I) and matrix catabolism (MMP-1 and MMP-13) genes even in the absence of the interleukins. Furthermore, the 3D bioprinted co-culture model displayed an upper hand in stimulating cartilage regeneration and OA inhibition than the spheroid model, underlining the role of silk fibroin-gelatin in encouraging chondrogenesis. Additionally, the 3D bioprinted silk-gelatin constructs further supported the maintenance of stable anti-inflammatory phenotype of M2 macrophage. Thus, the direct interaction between the primary OAC and M2 macrophages in the 3D context, along with the release of the soluble anti-inflammatory factors by the M2 cells, significantly contributed to a better understanding of the molecular mechanisms responsible for immune cell-mediated OA healing.

BMP signaling: A significant player and therapeutic target for osteoarthritis.

OBJECTIVE: To explore the significance of BMP signaling in osteoarthritis (OA) etiology, and thereafter propose a disease-modifying therapy for OA. METHODS: To examine the role of the BMP signaling in pathogenesis of OA, an Anterior Cruciate Ligament Transection (ACLT) surgery was performed to incite OA in C57BL/6J mouse line at postnatal day 120 (P120). Thereafter, to investigate whether activation of BMP signaling is necessary and sufficient to induce OA, we have used conditional gain- and loss-of-function mouse lines in which BMP signaling can be activated or depleted, respectively, upon intraperitoneal injection of tamoxifen. Finally, we locally inhibited BMP signaling through intra-articular injection of LDN-193189 pre- and post-onset surgically induced OA. The majority of the investigation has been conducted using micro-CT, histological staining, and immuno histochemistry to assess the disease etiology. RESULTS: Upon induction of OA, depletion of SMURF1-an intra-cellular BMP signaling inhibitor in articular cartilage coincided with the activation of BMP signaling, as measured by pSMAD1/5/9 expression. In mouse articular cartilage, the BMP gain-of-function mutation is sufficient to induce OA even without surgery. Further, genetic, or pharmacological BMP signaling suppression also prevented pathogenesis of OA. Interestingly, inflammatory indicators were also significantly reduced upon LDN-193189 intra-articular injection which inhibited BMP signaling and slowed OA progression post onset. CONCLUSION: Our findings showed that BMP signaling is crucial to the etiology of OA and inhibiting BMP signaling locally can be a potent strategy for alleviating OA.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies.

Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state (with the expression of collagen type X, matrix metalloproteinases-13, and alkaline phosphatase) analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause-effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies.

Rapid estimation of piperine in black pepper: Exploration of Raman spectroscopy.

INTRODUCTION: The major chemical marker of black pepper (Piper nigrum L) is piperine and its estimation is extremely important for quality assessment of black pepper. The methods for piperine quantification, to date, are laboratory based and use high end instruments like chromatographs, which require tedious sample processing and cause sample destruction. OBJECTIVES: In this article, we present a simple, rapid and green analytical method based on Raman spectroscopy for the quantitative assessment of piperine. MATERIAL AND METHODS: To assess the potential of the technique, we report the complete vibrational characterisation of the piperine with density functional theory (DFT) calculations. RESULTS: The theoretical peaks were obtained at 1097 cm-1 , 1388 cm-1 , 1528 cm-1 , 1578 cm-1 , and at 1627 cm-1 , and this result was verified in a Raman spectrometer followed by a preliminary experiment. Twenty black pepper samples were analysed using high-performance liquid chromatography (HPLC) and used as reference data for Raman analysis. The Raman shift spectra were analysed using partial least squares (PLS) and good prediction accuracy with correlation coefficient of prediction (Rp2 ) = 0.93, root mean square error of prediction (RMSEP) = 0.13 and residual prediction deviation (RPD) = 3.9 obtained. CONCLUSIONS: The results demonstrate the efficacy of the Raman technique for the estimation of piperine in the dry fruit of Piper nigrum.

Estimation of Andrographolides and Gradation of Andrographis paniculata Leaves Using Near Infrared Spectroscopy Together With Support Vector Machine.

Andrographis paniculata (Burm. F) Nees, has been widely used for upper respiratory tract and several other diseases and general immunity for a historically long time in countries like India, China, Thailand, Japan, and Malaysia. The vegetative productivity and quality with respect to pharmaceutical properties of Andrographis paniculata varies considerably across production, ecologies, and genotypes. Thus, a field deployable instrument, which can quickly assess the quality of the plant material with minimal processing, would be of great use to the medicinal plant industry by reducing waste, and quality grading and assurance. In this paper, the potential of near infrared reflectance spectroscopy (NIR) was to estimate the major group active molecules, the andrographolides in Andrographis paniculata, from dried leaf samples and leaf methanol extracts and grade the plant samples from different sources. The calibration model was developed first on the NIR spectra obtained from the methanol extracts of the samples as a proof of concept and then the raw ground samples were estimated for gradation. To grade the samples into three classes: good, medium and poor, a model based on a machine learning algorithm - support vector machine (SVM) on NIR spectra was built. The tenfold classification results of the model had an accuracy of 83% using standard normal variate (SNV) preprocessing.

Self-Assembling Nano-Globular Peptide from Human Lactoferrin Acts as a Systemic Enhancer of Bone Regeneration: A Novel Peptide for Orthopedic Application.

A technology for systemic and repeated administration of osteogenic factors for orthopedic use is an unmet medical need. Lactoferrin (∼80 kDa), present in milk, is known to support bone growth. We discovered a lactoferrin-mimetic peptide, LP2 (an 18-residue fragment from the N-terminus of the N-lobe of human lactoferrin), which self-assembles into a nano-globular assembly with a ß-sheet structure in an aqueous environment. LP2 is non-hemolytic and non-cytotoxic against human red blood cells and 3T3 fibroblasts, respectively, and appreciably stable in the human serum. LP2 through the bone morphogenetic protein-dependent mechanism stimulates osteoblast differentiation more potently than the full-length protein as well as the osteoblastic production of osteoprotegerin (an anti-osteoclastogenic factor). Consequently, daily subcutaneous administration of LP2 to rats and rabbits with osteotomy resulted in faster bone healing and stimulated bone formation in rats with a low bone mass more potently than that with teriparatide, the standard-of-care osteogenic peptide for osteoporosis. LP2 has skeletal bioavailability and is safe at the 15× osteogenic dose. Thus, LP2 is a novel peptide that can be administered systemically for the medical management of hard-to-heal fractures.

Simultaneous differentiation of articular and transient cartilage: WNT-BMP interplay and its therapeutic implication.

Limb skeleton forms through the process of endochondral ossification. This process of osteogenesis proceeds through an intermediate cartilage template and involves several stages of chondrocyte maturation and eventual bone formation. During the process of endochondral ossification, interplay between BMP and WNT signaling regulate simultaneous differentiation of articular and transient cartilage. In this review, we focus on the recent literature which explores the simultaneous differentiation of these two different types of cartilage. We discuss a new paradigm of developmental biology-inspired tissue engineering of bone and cartilage grafts and provide novel insight into treatment of osteoporosis.

Re-examining osteoarthritis therapy from a developmental biologist's perspective.

Osteoarthritis is the most prevalent musculoskeletal disorder and one for which there is no disease modifying therapy available at present. Our current understanding of the disease mechanism of osteoarthritis is limited owing to a lacuna of knowledge about the development and maintenance of articular cartilage that is affected during osteoarthritis. All current therapeutic strategies aim at countering inflammation which though mitigates pain but does not arrest the progressive degeneration of articular cartilage. During osteoarthritis, articular cartilage expresses markers for transient cartilage differentiation. Moreover, blocking transient cartilage differentiation is sufficient for halting the progression of experimental osteoarthritis. A developmental biology inspired approach that combines restoration of tissue microenvironment, supplementation with engineered cartilage and built in mechanism to prevent transient cartilage differentiation could be an avenue for developing a disease modifying therapy for osteoarthritis.

Investigating the mechanistic basis of biomechanical input controlling skeletal development: exploring the interplay with Wnt signalling at the joint.

Embryo movement is essential to the formation of a functional skeleton. Using mouse and chick models, we previously showed that mechanical forces influence gene regulation and tissue patterning, particularly at developing limb joints. However, the molecular mechanisms that underpin the influence of mechanical signals are poorly understood. Wnt signalling is required during skeletal development and is altered under reduced mechanical stimulation. Here, to explore Wnt signalling as a mediator of mechanical input, the expression of Wnt ligand and Fzd receptor genes in the developing skeletal rudiments was profiled. Canonical Wnt activity restricted to the developing joint was shown to be reduced under immobilization, while overexpression of activated ß-catenin following electroporation of chick embryo limbs led to joint expansion, supporting the proposed role for Wnt signalling in mechanoresponsive joint patterning. Two key findings advance our understanding of the interplay between Wnt signalling and mechanical stimuli: first, loss of canonical Wnt activity at the joint shows reciprocal, coordinated misregulation of BMP signalling under altered mechanical influence. Second, this occurs simultaneously with increased expression of several Wnt pathway component genes in a territory peripheral to the joint, indicating the importance of mechanical stimulation for a population of potential joint progenitor cells.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.

BMP signaling-driven osteogenesis is critically dependent on Prdx-1 expression-mediated maintenance of chondrocyte prehypetrophy.

During endochondral ossification, cartilage template is eventually replaced by bone. This process involves several well characterized, stereotypic, molecular and cellular changes in the cartilage primordia. These steps involve transition from resting to proliferative and then pre-hypertrophic to finally hypertrophic cartilage. BMP signaling is necessary and sufficient for osteogenesis. However, the specific step(s) of endochondral ossification in which BMP signaling plays an essential role is not yet known. In this study we have identified Prdx1, a known scavenger of ROS, to be expressed in pre-hypertrophic chondrocytes in a BMP signaling-dependent manner. We demonstrate that BMP signaling inhibition increases ROS levels in osteogenic cells. Further, Prdx1 regulates osteogenesis in vivo by helping maintenance of Ihh expressing pre-hypertrophic cells, in turn regulating these cells' transition into hypertrophy. Therefore, our data suggests that one of the key roles of BMP signaling in endochondral ossification is to maintain pre-hypertrophic state.

Precise spatial restriction of BMP signaling in developing joints is perturbed upon loss of embryo movement.

Dynamic mechanical loading of synovial joints is necessary for normal joint development, as evidenced in certain clinical conditions, congenital disorders and animal models where dynamic muscle contractions are reduced or absent. Although the importance of mechanical forces on joint development is unequivocal, little is known about the molecular mechanisms involved. Here, using chick and mouse embryos, we observed that molecular changes in expression of multiple genes analyzed in the absence of mechanical stimulation are consistent across species. Our results suggest that abnormal joint development in immobilized embryos involves inappropriate regulation of Wnt and BMP signaling during definition of the emerging joint territories, i.e. reduced ß-catenin activation and concomitant upregulation of pSMAD1/5/8 signaling. Moreover, dynamic mechanical loading of the developing knee joint activates Smurf1 expression; our data suggest that Smurf1 insulates the joint region from pSMAD1/5/8 signaling and is essential for maintenance of joint progenitor cell fate.

Developmental Biology-Inspired Strategies To Engineer 3D Bioprinted Bone Construct.

A major challenge in bone tissue engineering is to develop clinically conformant load-bearing bone constructs in a patient-specific manner. A paradigm shift would involve combination of developmental engineering and 3D bioprinting to optimize strategies focusing on close simulation of in vivo developmental processes using in vitro tissue engineering approaches. This study demonstrates that silk-gelatin bioink could activate the canonical Wnt/ß-catenin and Indian hedgehog (IHH) pathways during osteogenic differentiation of mesenchymal stem cells (TVA-BMSC), laden in 3D bioprinted constructs. Temporal gene expression related to early and terminal osteogenic differentiation of the TVA-BMSC in 3D bioprinted constructs closely followed the in vivo processes. This was evidenced by expression of early differentiation markers (RUNX2 and COL I), mid- and mid-to-late-stage markers (ALP, ON, OPN, and OCN), and terminal osteocytic genes (PDPN, DMP1, and SOST). Furthermore, a combinatorial effect of addition of T3 and simulation of the endochondral ossification route could activate the parathyroid hormone (PTH), IHH, and Wnt/ß-catenin pathways, thus improving the osteogenic differentiation potential of stem cells and improved mineralization. The endochondral ossification observed in vitro in our study shows stark similarities to in vivo endochondral ossification-based limb skeletal development, specifically (1) chondrogenic condensation and hypertrophic cartilaginous template development, (2) involvement of IHH signaling indicative of the development of bony collar by perichondral ossification, (3) involvement of Wnt/ß-catenin signaling, (4) involvement of PTH signaling, and (5) synthesis and deposition of bone-specific mineral. Thus, induction of differentiation of progenitor cells to osteoblasts in 3D bioprinted constructs, while recapitulating the developmental-biology-inspired endochondral ossification route, may offer an important therapeutic proposition to develop clinically conformant bone construct.

NFIA and GATA3 are crucial regulators of embryonic articular cartilage differentiation.

During appendicular skeletal development, the bi-potential cartilage anlagen gives rise to transient cartilage, which is eventually replaced by bone, and to articular cartilage that caps the ends of individual skeletal elements. While the molecular mechanism that regulates transient cartilage differentiation is relatively well understood, the mechanism of articular cartilage differentiation has only begun to be unraveled. Furthermore, the molecules that coordinate the articular and transient cartilage differentiation processes are poorly understood. Here, we have characterized in chick the regulatory roles of two transcription factors, NFIA and GATA3, in articular cartilage differentiation, maintenance and the coordinated differentiation of articular and transient cartilage. Both NFIA and GATA3 block hypertrophic differentiation. Our results suggest that NFIA is not sufficient but necessary for articular cartilage differentiation. Ectopic activation of GATA3 promotes articular cartilage differentiation, whereas inhibition of GATA3 activity promotes transient cartilage differentiation at the expense of articular cartilage. We propose a novel transcriptional circuitry involved in embryonic articular cartilage differentiation, maintenance and its crosstalk with the transient cartilage differentiation program.

BMP signaling is required for adult skeletal homeostasis and mediates bone anabolic action of parathyroid hormone.

Bmp2 and Bmp4 genes were ablated in adult mice (KO) using a conditional gene knockout technology. Bones were evaluated by microcomputed tomography (µCT), bone strength tester, histomorphometry and serum biochemical markers of bone turnover. Drill-hole was made at femur metaphysis and bone regeneration in the hole site was measured by calcein binding and µCT. Mice were either sham operated (ovary intact) or ovariectomized (OVX), and treated with human parathyroid hormone (PTH), 17ß-estradiol (E2) or vehicle. KO mice displayed trabecular bone loss, diminished osteoid formation and reduced biomechanical strength compared with control (expressing Bmp2 and Bmp4). Both osteoblast and osteoclast functions were impaired in KO mice. Bone histomorphomtery and serum parameters established a low turnover bone loss in KO mice. Bone regeneration at the drill-hole site in KO mice was lower than control. However, deletion of Bmp2 gene alone had no effect on skeleton, an outcome similar to that reported previously for deletion of Bmp4 gene. Both PTH and E2 resulted in skeletal preservation in control-OVX, whereas in KO-OVX, E2 but not PTH was effective which suggested that the skeletal action of PTH required Bmp ligands but E2 did not. To determine cellular effects of Bmp2 and Bmp4, we used bone marrow stromal cells in which PTH but not E2 stimulated both Bmp2 and Bmp4 synthesis leading to increased Smad1/5 phosphorylation. Taken together, we conclude that Bmp2 and Bmp4 are essential for maintaining adult skeletal homeostasis and mediating the anabolic action of PTH.

Characterization of BMP signaling dependent osteogenesis using a BMP depletable avianized bone marrow stromal cell line (TVA-BMSC).

Adipogenesis, chondrogenesis and osteogenesis are BMP signaling dependent differentiation processes. However, the molecular networks operating downstream of BMP signaling to bring about these distinct fates are yet to be fully elucidated. We have developed a novel Bone Marrow Stromal Cell (BMSC) derived mouse cell line as a powerful in vitro platform to conduct such experiments. This cell line is a derivative of BMSCs isolated from a tamoxifen inducible Bmp2 and Bmp4 double conditional knock-out mouse strain. These BMSCs are immortalized and stably transfected with avian retroviral receptor TVA (TVA-BMSCs), enabling an easy method for stable transduction of multiple genes in these cells. In TVA-BMSCs multiple components of BMP signaling pathway can be manipulated simultaneously. Using this cell line we have demonstrated that for osteogenesis, BMP signaling is required only for the first three days. We have further demonstrated that Klf10, an osteogenic transcription factor which is transcribed in developing bones in a BMP signaling dependent manner, can largely compensate for the loss of BMP signaling during osteogenesis of BMSCs. TVA-BMSCs can undergo chondrogenesis and adipogenesis, and hence may be used for dissection of the molecular networks downstream of BMP signaling in these differentiation processes as well.