Comparing the bone regeneration potential between a trabecular bone and a porous scaffold through osteoblast migration and differentiation: A multiscale approach.

Both cell migration and osteogenic differentiation are critical for successful bone regeneration. Therefore, understanding the mechanobiological aspects that govern these two processes is essential in designing effective scaffolds that promote faster bone regeneration. Studying these two factors at different locations is necessary to manage bone regeneration in various sections of a scaffold. Hence, a multiscale computational model was used to observe the mechanical responses of osteoblasts placed in different positions of the trabecular bone and gyroid scaffold. Fluid shear stresses in scaffolds at cell seeded locations (representing osteogenic differentiation) and strain energy densities in cells at cell substrate interface (representing cell migration) were observed as mechanical response parameters in this study. Comparison of these responses, as two critical factors for bone regeneration, between the trabecular bone and gyroid scaffold at different locations, is the overall goal of the study. This study reveals that the gyroid scaffold exhibits higher osteogenic differentiation and cell migration potential compared to the trabecular bone. However, the responses in the gyroid only mimic the trabecular bone in two out of nine positions. These findings can guide us in predicting the ideal cell seeded sites within a scaffold for better bone regeneration and in replicating a replaced bone condition by altering the physical parameters of a scaffold.

Shelf life enhancement technique of Musa acuminata in a controlled environment and optimization of process parameters affecting shelf life using genetic algorithm.

An economical and effective storage solution has been designed in this work for the storage of postharvest fruits and vegetables. Musa acuminata or banana has a shelf life of 5-6 days in open uncontrolled environment. This article reports a storage solution of M. acuminata in a controlled enclosure containing titanium oxide (TiO2 )-coated inner walls and irradiated with ultraviolet ray of band "C," an air filtration unit, 5% by volume potassium permanganate (KMnO4 ) solution in a clay pot, grow lights, and activated charcoal granules. The same fruit was kept in an uncontrolled environment too. The percentages of dark spots on banana (M. acuminata) upon storage in controlled and uncontrolled environments have been estimated using an image-processing algorithm. The prediction of dark spots was conducted using multi-linear and multivariate polynomial regression. Experimentation with optimum process parameters obtained with genetic algorithm resulted in a shelf life extension of 6 days as compared to its storage in an uncontrolled environment. The setup can be used in vegetable and fruit markets for the extension of shelf life of postharvest perishable items in a compact and cost-effective manner. The setup does not use any refrigeration process thereby decreasing energy requirement.

Chirality-induced Lineage Enforcement of Mechanosensitive Mesenchymal Stem Cells Across Germ Layer Boundaries.

Mesenchymal to epithelial transition (MET) is instrumental in embryogenesis, tissue repair, and wound healing while the epithelial to mesenchymal transition (EMT) plays role in carcinogenesis. Alteration in microenvironment can modulate cellular signaling and induce EMT and MET. However, modulation of microenvironment to induce MET has been relatively less explored. In this work, effect of matrix stiffness in mediating MET in umbilical cord-derived mesenchymal stem cells (UCMSC) is investigated. Differential segregation of cell fate determinant proteins is one of the key factors in mediating altered stem cell fates through MET even though the genesis of apicobasal polarity remains ambiguous. Herein, it is also attempted to decipher if microenvironment-induced asymmetric cell division has a role to play in driving the cells toward MET. UCMSC cultured on stiffer PDMS matrices resulted in significantly (p < 0.05) higher expression of mechanotransduction proteins. It was also observed that stiffer matrices mediated significant (p < 0.05) upregulation of the polarity proteins and cell fate determinant protein, and epithelial marker proteins over lesser stiff substrates. On the contrary, expression of inflammatory and mesenchymal markers was reduced significantly (p < 0.05) on the stiffer matrices. Cell cycle analysis showed a significant increase in the G1 phase among the cells seeded on stiffer matrices. Transcriptomic studies validated higher expression of epithelial markers genes and lower expression of EMT markers. The transition from mesenchymal to epithelial phenotype depending on the gradation in matrix stiffness is successfully demonstrated. A computational machine learning model was developed to validate stiffness-MET correlation with 94% accuracy. The cross-boundary trans-lineage differentiation capability of MSC on bioengineered substrates can be used as a potential tool in tissue regeneration, organogenesis, and wound healing applications. In our present study, we deciphered the correlation between YAP/TAZ mechanotransduction pathway, EMT signaling pathway, and asymmetric cell division in mediating MET in MSC in a substrate stiffness-dependent manner. It is inferred that the stiffer PDMS matrices facilitate the transition from mesenchymal to epithelial state of MSC. Further, our study also proposed a scoring system to sort MSC from an intermediate hybrid E/M population while undergoing graded MET on matrices of different stiffnesses using a machine learning technique. This proposed scoring system can provide information regarding the E/M state of MSC on different bioengineered constructs based on their biophysical properties which may help in the proper choice of biomaterials in complex tissue-engineering applications.

In-silico study of type 'B' condylar head fractures and evaluating the influence of two positional screw distance in two-screw osteosynthesis construct.

Clinical fixation screws are common in clinical practices to fix mandibular condyle fractures. Evidence suggests significance of 'working length' that is, distance between proximal and distal fixation screws in proximity to the fracture in orthopaedic implant design. In pursuit of stable implant-bone construct, this study aims to investigate the biomechanical performance of each configuration considered in the study and provide an optimal working length between the screws for clinical reference. Finite element models of virtually designed broken condyle as type 'B' were simulated and analysed in ANSYS Workbench. Screws are implanted according to previous literature at five varied distances 'd' maintaining five different ratios with the fracture length 'D'. Based on a literature review, boundary conditions, muscle traction forces and non-linear contacts were assigned to obtain precise results. Each case is considered an individual configuration and von Mises distribution, microstrain in bone, screw-bone interface micromotion and fracture dislocation were evaluated for all these configurations. Stress-shielding phenomenon is observed for maximum von Mises stresses in bone. Microstrain concentration was significant in cancellous bone in the vicinity of the screw around the fracture line. Configurations were compared based on the stress-strain along with micromotion to support the required amount of osseointegration between implant and bone. Presented data from all five conditions supported the assumption that under physiological loading conditions, the D3 configuration provided stability for fracture healing. Further research on screw shapes, diameters and material properties, or investigating the direction of forces within the screws could provide further insight into this topic.

Design and development of a patient-specific temporomandibular joint implant: Probing the influence of bone condition on biomechanical response.

Total temporomandibular joint (TMJ) replacement is widely recognized as an effective treatment for TMJ disorders. The long-term stability of TMJ implants depends on two important factors which are design concepts for fixation to anatomical locations in the mandible and bone conditions. Other factors include stress distribution, microstrain in the peri-implant, bone attributes like bone conditions leading to the clinical complications and failures. This study addresses these limitations by examining the influence of patient-specific design concepts and bone conditions on TMJ implant performance. Clinical evidences support the importance of implant design on healing ability. Previous studies have focused on achieving precise implant fit based on geometric considerations, however those published studies did not explore the impact of such. Against this perspective, the present study reports the extensive finite element analysis (FEA) results, while analyzing the impact of a newly designed patient-specific TMJ implant to address clinical complications associated with various bone conditions, particularly osteoporotic bone. In validating the FEA results, the performance of additively manufactured patient-specific TMJ implants was compared with designs resembling two commonly used clinically approved implant designs. By addressing the limitations of previous research and emphasizing the importance of bone conditions, the study provides valuable guidelines for the development of next-generation TMJ implants. These findings contribute to enhanced clinical outcomes and long-term success in the treatment of TMJ disorders.

Biomechanical analysis of the novel S-type dynamic cage by implementation of teaching learning based optimization algorithm - An experimental and finite element study.

Anterior Cervical Discectomy and Fusion (ACDF) is the most popular and effective procedure for patients with intervertebral disc degeneration, where the degenerated disc is replaced with an interbody implant (widely known as cage). The design of the cage plays a vital role since it has to provide stability for the anterior cervical column without any side-effects. We designed a novel S-type dynamic cage for C4-C5 level, using Polyetheretherketone (PEEK) material considering four different shapes namely: square, circle, rectangle and elliptical, for the central window to occupy bone graft. The major design constrain for a successful cage is minimized cage stress, in order to avoid subsidence. Finite Element (FE) analysis results revealed that the cage stress values obtained during the physiological motion varied depending upon the shape of the central window provided for bone graft. The objective of this study is to optimize the central window shape using the Teaching Learning Based Optimization (TLBO) algorithm. It was found that square and elliptical shape bone graft cavity resulted in better outcomes. Additional experimental study was also conducted with a six-axis spine simulator. Based on the optimization results, we manufactured two PEEK cage models with square and elliptical shaped central window using additive manufacturing. A prototype model of the C4-C5 level made of Polyvinylchloride (PVC) was used for experiment due to the existing constraints for using a cadaveric model. The experimental results were cross-verified using FE analysis. Thus, we would like to conclude that square and elliptical shape of the central window were the better design factor for our novel dynamic cage.

Modelling cell deformations in bioprinting process using a multicompartment-smooth particle hydrodynamics approach.

Bioprinting using cell-laden bioink is a rapidly emerging additive manufacturing method to fabricate engineered tissue constructs and in vitro models of disease biology. Amongst different bioprinting modalities, extrusion-based bioprinting is the most conveniently adopted technique due to its affordability. Bioinks consisting of living cells are suspended in hydrogels and extruded through syringe-needle assemblies, which subsequently undergo gelation at the collector plate. During the process, pressure is exerted on living cells which may cause cell deaths. Thus, for selected combination of cell and hydrogel, exerted pressure and the extrusion play key roles in determining the cell viability. Experimental evaluation to characterise stresses experienced by the cells in a bioink during bioprinting is a tedious exercise. Herein, computational modelling can be applied efficiently for rapid screening of bioinks. In the present study, a smoothed particle hydrodynamics model is developed for the analysis of stresses exerted on the cells during bioprinting process. Cells are modelled by assigning different mechanical properties to nucleus, cytoskeleton and cell membrane regions of the cell to get a more realistic understanding of cell deformation. The cytoplasm and nucleus are modelled as finite element meshes and a spring model of the cell membrane is coupled to the finite element model to develop a three-compartment model of the cell. Cell deformation is taken as a potential indicator of cell death. Effect of different process parameters such as flow rate, syringe-nozzle geometry and cell density are investigated. A submodeling approach is further introduced to predict deformation with higher resolution in a unit volume containing 104 to 108 cells. Results suggest that the generated bioink flow dynamic model can be a useful tool for the computational study of fluid flow involving cell suspensions during a bioprinting process.

Leveraging Substrate Stiffness to Promote Stem Cell Asymmetric Division via Mechanotransduction-Polarity Protein Axis and Its Bayesian Regression Analysis.

Asymmetric division of stem cells is an evolutionarily conserved process in multicellular organisms responsible for maintaining cellular fate diversity. Symmetric-asymmetric division pattern of mesenchymal stem cells (MSCs) is regulated by both biochemical and biophysical cues. However, modulation of mechanotransduction pathway by varying scaffold properties and their adaptation to control stem cell division fate is not widely established. In this study, we explored the interplay between the mechanotransduction pathway and polarity protein complex in stem cell asymmetry under varied biophysical stimuli. We hypothesize that variation of scaffold stiffness will impart mechanical stimulus and control the cytoskeleton assembly through RhoA, which will lead to further downstream activation of polarity-related cell signaling and asymmetric division of MSCs. To establish the hypothesis, umbilical cord-derived MSCs were cultured on polycaprolactone/collagen scaffolds with varied stiffness, and expression levels of several important genes (viz., Yes-associated protein [YAP], transcriptional coactivator with PDZ-binding motif [TAZ], LATS1, LATS2, Par3, Par6, PRKC1 [homolog of aPKC] and RhoA), and biomarkers (viz. YAP, TAZ, F-actin, Numb) were assessed. Support vector machine polarity index was employed to understand the polarization status of the MSCs cultured on varied scaffold stiffness. Furthermore, the Bayesian logistic regression model was employed for classifying the asymmetric division of MSCs cultured on different scaffold stiffnesses that showed 91% accuracy. This study emphasizes the vital role of scaffold properties in modulating the mechanotransduction signaling pathway of MSCs and provides mechanistic basis for adopting facile method to control stem cell division pattern toward improving tissue engineering outcome.

Oxidative Damage Induced Telomere Mediated Genomic Instability in Cells from Ataxia Telangiectasia Patients.

Our cellular genome is susceptible to cytotoxic lesions which include single strand breaks and double strand breaks among other lesions. Ataxia telangiectasia mutated (ATM) protein was one of the first DNA damage sensor proteins to be discovered as being involved in DNA repair and as well as in telomere maintenance. Telomeres help maintain the stability of our chromosomes by protecting the ends from degradation. Cells from ataxia telangiectasia (AT) patients lack ATM and accumulate chromosomal alterations. AT patients display heightened susceptibility to cancer. In this study, cells from AT patients (called as AT -/- and AT +/- cells) were characterized for genome stability status and it was observed that AT -/- cells show considerable telomere attrition. Furthermore, DNA damage and genomic instability were compared between normal (AT +/+ cells) and AT -/- cells exhibiting increased frequencies of spontaneous DNA damage and genomic instability markers. Both AT -/- and AT +/- cells were sensitive to sodium arsenite (1.5 and 3.0 µg/ml) and ionizing radiation-induced (2 Gy, gamma rays) oxidative stress. Interestingly, telomeric fragments were detected in the comet tails as revealed by comet-fluorescence in situ hybridization analysis, suggestive of telomeric instability in AT -/- cells upon exposure to sodium arsenite or radiation. Besides, there was an increase in the number of chromosome alterations in AT -/- cells following arsenite treatment or irradiation. In addition, complex chromosome aberrations were detected by multicolor fluorescence in situ hybridization in AT -/- cells in comparison to AT +/- and normal cells. Telomere attrition and chromosome alterations were detected even at lower doses of sodium arsenite. Peptide nucleic acid - FISH analysis revealed defective chromosome segregation in cells lacking ATM proteins. The data obtained in this study substantiates the role of ATM in telomere stability under oxidative stress.

Finite element analysis of the influence of cyclic strain on cells anchored to substrates with varying properties.

The response of cytoskeleton to mechanical cues plays a pivotal role in understanding several aspects of cellular growth, migration, and cell-cell and cell-matrix interactions under normal and diseased conditions. Finite element analysis (FEA) has become a powerful computational technique to study the response of cytoskeleton in the maintenance of overall cellular mechanics. With the revelation of role of external mechanical microenvironment on cell mechanics, FEA models have also been developed to simulate the effect of substrate stiffness on the mechanical properties of cancer cells. However, the models developed so far model cellular response under static mode, whereas in physiological condition, cells always experience dynamic loading conditions. To develop a more accurate model of cell-extracellular matrix (ECM) interactions, this paper models the cytoskeleton and other parts of the cell by beam and solid elements respectively, assuming spherical morphology of the cell. The stiffness and roughness of extracellular matrix were varied. Furthermore, static and dynamic sinusoidal loads were applied through a flat plate indenter on the cell along with providing sinusoidal strain at the substrate. It is observed that due to axial loading, cell reaches a plastic region, and when the sinusoidal loading is added to the axial load, the cell experiences permanent deformation. Degradation of the cytoskeleton elements and a physiologically more relevant spherical cap shape of the cell were also considered during the analysis. This study suggests that asperity topology of the substrate and indirect cyclic load can play a significant role in the shape alterations and motion of a cell.

Assessment of Jaw Bone Quality for Designing Patient-Specific Dental Implant Using Computed Tomography Data.

It is essential to assess bone density among mandible teeth as well as among patients and also to observe the variation in all mechanical parameters of the bone for accurate design of patient-specific dental implants. This information helps in the design of implants to create a more osseointegration-friendly environment at the bone adjacent to the implant. For this study, 40 patients were chosen irrespective of age, sex, and bone density. Hounsfield Units were calculated using cone beam computed tomography data. Seven teeth were studied: central incisor, lateral incisor, canine, first premolar, second premolar, first molar, and second molar. A total of 12 arbitrary points were chosen in both buccal and lingual sides which were further divided into external and internal. From the analysis, it was observed that the bone density of the central incisor and that of the canine is greater than that of the molars.

Substrate-dependent control of the chiral orientation of mesenchymal stem cells: image-based quantitative profiling.

Stem-cell (SC) chirality or left-right (LR) asymmetry is an essential attribute, observed during tissue regeneration. The ability to control the LR orientation of cells by biophysical manipulation is a promising approach for recapitulating their inherent function. Despite remarkable progress in tissue engineering, the development of LR chirality in SCs has been largely unexplored. Here, we demonstrate the role of substrate stiffness on the LR asymmetry of cultured mesenchymal stem cells (MSCs). We found that MSCs acquired higher asymmetricity when cultured on stiffer PCL/collagen matrices. To confirm cellular asymmetry, different parameters such as the aspect ratio, orientation angle and intensity of polarized proteins (Par) were investigated. The results showed a significant (p < 0.01) difference in the average orientation angle, the cellular aspect ratio, and the expression of actin and Par proteins in MSCs cultured on matrices with different stiffnesses. Furthermore, a Gaussian support-vector machine was applied to classify cells cultured on both (2% and 10% PCL/Collagen) matrices, with a resulting accuracy of 96.2%. To the best of our knowledge, this study is the first that interrelates and quantifies MSC asymmetricity with matrix properties using a simple 2D model.

Bioprinting of radiopaque constructs for tissue engineering and understanding degradation behavior by use of Micro-CT.

Bioprinting has emerged as a potential technique to fabricate tissue engineering constructs and in vitro models directly using living cells as a raw material for fabrication, conforming to the heterogeneity and architectural complexity of the tissues. In several of tissue engineering and in vitro disease modelling or surgical planning applications, it is desirable to have radiopaque constructs for monitoring and evaluation. In the present work, enhanced radiopaque constructs are generated by substituting Calcium ions with Barium ions for crosslinking of alginate hydrogels. The constructs are characterized for their structural integrity and followed by cell culture studies to evaluate their biocompatibility. This was followed by the radiopacity evaluation. The radiological images obtained by micro-CT technique was further applied to investigate the degradation behavior of the scaffolds. In conclusion, it is observed that barium crosslinking can provide a convenient means to obtain radiopaque constructs with potential for multi-faceted applications.

Clinico-pathological significance of Drp1 dysregulation and its correlation to apoptosis in oral cancer patients.

Dysregulation in mitochondrial dynamics has been associated with several diseases including cancer. Present study assessed the alteration in mitochondrial fission protein (Drp1) in oral epithelial cells collected from clinically confirmed pre-cancer and cancer patients and further correlates it with the cellular apoptosis signaling. Results indicate the ROS accumulation in OSCC patients is accompanied by several changes including increase in mitochondrial mass, expression of mitochondrial fission protein (Drp1) and alteration in apoptotic signaling. The positive co-relation has been observed between the expressions of anti-apoptotic Bcl-2proteinswith mitochondrial fission protein Drp1. Higher mitochondrial fission in oral cancer cells was also correlated with the increased expression of cell cycle marker CyclinD1 indicating highly proliferative stage of oral cancer cells. The clinical correlation can be extended to develop biomarker for diagram and program in oral cancer management.

α-Lipoic acid prevents the ionizing radiation-induced epithelial-mesenchymal transition and enhances the radiosensitivity in breast cancer cells.

Radiotherapy is routinely used in the treatment of breast cancer. However, its efficiency is often limited by the development of radioresistance and metastasis. The cancer cells surviving irradiation show epithelial-mesenchymal transition (EMT) along with increased migration, invasion and metastasis. In this study, we have evaluated the role of α-lipoic acid in preventing the radiation-induced EMT and in sensitizing the breast cancer cells to radiation. The breast cancer cell lines, MCF-7 and MDA-MB-231 were pretreated with lipoic acid, irradiated and the changes associated with cell growth, clonogenicity, migration, matrix metalloproteinases (MMPs), EMT and TGFß signaling were measured. Our data showed that lipoic acid pretreatment sensitized the breast cancer cells to the ionizing radiation and inhibited the radiation-induced migration and the release of MMP2 and MMP9. Lipoic acid also prevented the TGFß1 release and inhibited the radiation-induced EMT in breast cancer cells. The inhibition of TGFß signaling by lipoic acid is associated with the inhibition of radiation-induced activation and translocation of NF-κB. These results suggest that α-lipoic acid inhibits the radiation-induced TGFß signaling and nuclear translocation of NF-κB, thereby inhibiting the radiation-induced EMT and sensitizing the breast cancer cells to ionizing radiation.

Role of telomeric RAP1 in radiation sensitivity modulation and its interaction with CSC marker KLF4 in colorectal cancer.

Aims: Radiotherapy is predominantly used as one of the treatment modalities to treat local tumor in colorectal cancer (CRC). Hindrance in disease treatment can be attributed to radio-tolerance of cancer stem cells (CSCs) subsistence in the tumor. Understanding the radio-resistant property of CSCs might help in the accomplishment of targeted radiotherapy treatment and increased disease-free survival. Telomeric RAP1 contributes in modulation of various transcription factors leading to aberrant cell proliferation and tumor cell migration. Therefore, we investigated the role of RAP1 in maintaining resistance phenotype and acquired stemness in radio-resistant cells.Main methods: Characterization of HCT116 derived radio-resistant cell (HCT116RR) was performed by cell survival and DNA damage profiling. RAP1 silenced cells were investigated for DNA damage and expression of CSC markers through western blotting and Real-time PCR post-irradiation. Molecular docking and co-immunoprecipitation study were performed to investigate RAP1 and KLF4 interaction followed by RAP1 protein status profiling in CRC patient.Key findings: We established radio-resistant cells, which showed tolerance to radiotherapy and elevated expression of CSC markers along with RAP1. RAP1 silencing showed enhanced DNA damage and reduced expression of CSC markers post-irradiation. We observed strong physical interaction between RAP1 and KLF4 protein. Furthermore, higher RAP1 expression was observed in the tumor of CRC patients. Dataset analysis also revealed that high expression of RAP1 expression is associated with poor prognosis.Significance: We conclude that higher expression of RAP1 implicates its possible role in promoting radio-resistance in CRC cells by modulating DNA damage and CSC phenotype.

Honey-incorporated nanofibre reduces replicative senescence of umbilical cord-derived mesenchymal stem cells.

Umbilical cord-derived mesenchymal stem cells (UCDMSC) are attractive candidates for cell-based regenerative medicine. However, they are susceptible to replicative senescence during repetitive passaging for in-vitro expansion and induced senescence in an oxidative, inflammatory microenvironment in vivo. Aim of this study is to investigate if honey-incorporated matrices can be employed to reduce senescence of UCDMSC. Matrices were prepared by electrospinning solutions of honey with poly-vinyl alcohol (PVA). PVA:honey matrices exhibited free radical scavenging activity. Culture of UCDMSC on PVA:honey matrices showed improvement in cell proliferation compared to pure PVA nanofibres. Expression of vimentin indicated that mesenchymal phenotype is preserved after culturing on these matrices. Further, UCDMSC were serially subcultured and cells of two passages (P2 and P6) were evaluated for reactive oxygen species (ROS) load and senescence parameters. P6 cells showed a higher ROS load and ß-galactosidase (ß-gal) positive senescent cells compared to P2. However, culturing on PVA:honey substrates significantly reduced both ROS and ß-gal markers compared to cells on PVA substrates. Honey contains several antioxidant and anti-inflammatory components, which can reduce the ROS-related senescence. Thus, it is concluded that honey containing nanofibres can be effective substrates for stem cell-based wound healing and regenerative medicine.

Mechanical response at peri-implant mandibular bone for variation of pore characteristics of implants: A Finite Element Study.

PURPOSE: In this paper, the mechanical response of generic dental implants having calculated porosities with varying pore-sizes has been evaluated. The purpose of this study was to compare the developed stress-strain of designed porous implants (i.e., stress at the implant and strain at the peri-implant bone) with that of the non-porous implant. METHODS: 3D model of a mandible was prepared from CT scan data and nine generic dental implant models have been designed having 10%, 20%, and 30% porosity with 500, 700, and 900 micron pore size along with a non-porous model for carrying out FE analyses. First, failure analyses of implants, under a biting force of 250 N have been performed. Next, the remaining implants have been further evaluated under average compressive chewing load of 100 N, for mechanical responses at bone-implant interface. RESULTS: Von Mises strain at the peri-implant mandibular bone increases with the increase in percentage porosity of the implant material and maximum implant stress remained much below the yield stress level. CONCLUSION: Implant stiffness and compressive strength vary as a function of porosity and pore size. Strain obtained on the peri-implant bone is sufficient enough to facilitate better bone growth with the 700 micron pore size and 30% porosity, thus reducing the effect of stress shielding.

Bioink formulations to ameliorate bioprinting-induced loss of cellular viability.

Extrusion bioprinting, the most affordable and convenient bioprinting modality, is also associated with high process-induced cell deaths. Mechanical stresses on the cells during pneumatic or piston extrusion generate excessive reactive oxygen species and activate apoptosis, inflammatory pathways in the cells. In this study, a bioink formulation is augmented with an antioxidant, N-acetyl cysteine (NAC) as a possible solution to abrogate the effect of bioprinting-associated cell survival losses. The NAC addition to bioinks did not affect the bioprinting process, shape fidelity, or the mechanical properties of the constructs to any large extent. However, the bioprinting process conducted at 0.30 MPa pressure and 410 µm nozzle inner diameter with bioinks of 3% w/v alginate, 105 cells/ml resulted in survival losses of up to 25% for MC3T3 cells. In contrast, NAC bioinks showed a significant (p < 0.01) improvement in day 1 cell survival (91%), while the enhancement in day 3 cell viability was still greater. It was further observed that the reactive oxygen species (ROS) load of bioprinted constructs was approximately 1.4 times higher compared to control, whereas NAC containing constructs reduced the ROS load at levels comparable to control samples. The effect on apoptosis and inflammation markers showed that NAC had a greater role in modulating apoptosis. It is concluded that the presented approach to preserve cell viability and functionality would be advantageous over other contemporary methods (like alterations in extrusion pressure, nozzle diameter, polymer concentration, etc.) as viability can be preserved without compromising the fabrication time or the resolution/mechanical properties of the constructs with this bioink formulation approach.

Isolation and mass spectrometry based hydroxyproline mapping of type II collagen derived from Capra hircus ear cartilage.

Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage. Delivery of COLII from animal sources could improve cartilage regeneration therapies. Here we show that COLII can be purified from the Capra ear cartilage, a commonly available bio-waste product, with a high yield. MALDI-MS/MS analysis evidenced post-translational modifications of the signature triplet, Glycine-Proline-Hydroxyproline (G-P-Hyp), in alpha chain of isolated COLII (COLIIA1). Additionally, thirty-two peptides containing 59 Hyp residues and a few G-X-Y triplets with positional alterations of Hyp in COLIIA1 are also identified. Furthermore, we show that an injectable hydrogel formulation containing the isolated COLII facilitates chondrogenic differentiation towards cartilage regeneration. These findings show that COLII can be isolated from Capra ear cartilage and that positional alteration of Hyp in its structural motif, as detected by newly developed mass spectrometric method, might be an early marker of cartilage disorder.