Modulating the hAM/PCL Biocomposite for Expedited Wound Healing: A Chemical-Free Approach for Boosting Regenerative Potential.

There is an arising need for effective wound dressings that retain the bioactivity of a cellular treatment, but without the high costs and complexities associated with manufacturing, storing, and applying cell-based products. As skin wound recovery is a dynamic and complicated process, a significant obstacle to the healing of skin wounds is the lack of an appropriate wound dressing that can imitate the microenvironment of healthy skin and prevent bacterial infection. It requires the well-orchestrated integration of biological and molecular events. In this study, we have fabricated full-thickness skin graft biocomposite membranes to target full-thickness skin excision wounds. We reinforced human amniotic membrane (hAM) with electrospun polycaprolactone (PCL) to develop composite membranes, namely, PCL/hAM and PCL/hAM/PCL. Composite membranes were compared for physical, biological, and mechanical properties with the native counterpart. PCL/hAM and PCL/hAM/PCL displayed improved stability and delayed degradation, which further synergically improved the rapid wound healing property of hAM, driven primarily by wound closure analysis and histological assessment. Moreover, PCL/hAM displayed a comparable cellular interaction to hAM. On application as a wound dressing, histological analysis demonstrated that hAM and PCL/hAM promoted early epidermis and dermis formation. Studies on in vivo wound healing revealed that although hAM accelerates cell development, the overall wound healing process is similar in PCL/hAM. This finding is further supported by the immunohistochemical analysis of COL-1/COL-3, CD-31, and TGF-ß. Overall, this conjugated PCL and hAM-based membrane has considerable potential to be applied in skin wound healing. The facile fabrication of the PCL/hAM composite membrane provided the self-regenerating wound dressing with the desired mechanical strength as an ideal regenerative property for skin tissue regeneration.

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.

Self-Cleavage of Human Chloride Channel Accessory 2 Causes a Conformational Shift That Depends on Membrane Anchorage and Is Required for Its Regulation of Store-Operated Calcium Entry.

Human CLCA2 regulates store-operated calcium entry (SOCE) by interacting with Orai1 and STIM1. It is expressed as a 943aa type I transmembrane protein that is cleaved at amino acid 708 to produce a diffusible 100 kDa product. The N-terminal ectodomain contains a hydrolase-like subdomain with a conserved HEXXH zinc-binding motif that is proposed to cleave the precursor autoproteolytically. Here, we tested this hypothesis and its link to SOCE. We first studied the conditions for autocleavage in isolated membranes and then in a purified protein system. Cleavage was zinc-dependent and abolished by mutation of the E in the HEXXH motif to Q, E165Q. Cleavage efficiency increased with CLCA2 concentration, implying that it occurs in trans. Accordingly, the E165Q mutant was cleaved by co-transfected wildtype CLCA2. Moreover, CLCA2 precursors with different epitope tags co-immunoprecipitated. In a membrane-free system utilizing immunopurified protease and target, no cleavage occurred unless the target was first denatured, implying that membranes provide essential structural or conformational cues. Unexpectedly, cleavage caused a conformational shift: an N-terminal antibody that immunoprecipitated the precursor failed to precipitate the N-terminal product unless the product was first denatured with an ionic detergent. The E165Q mutation abolished the stimulation of SOCE caused by wildtype CLCA2, establishing that the metalloprotease activity is required for this regulatory function.

Mechanistic crosstalk of extracellular calcium-mediated regulation of maturation and plasticity in human monocytes.

Innate immune cells play a pivotal role in controlling tissue repair and rejection after biomaterial implantation. Calcium supplementation regulates cellular responses and alter the pathophysiology of various diseases. A series of macrophage activations through differential plasticity has been observed after cell-to-material interactions. We investigated the role of calcium supplementation in controlling macrophage phenotypes in pro-inflammatory and pre-reparative states. Oxidative defence and mitochondria involvement in cellular plasticity and the sequential M0 to M1 and M1 to M2 transitions were observed after calcium supplementation. This study describes the molecular mechanism of reactive oxygen species and drives the interconnected cellular plasticity of macrophages in the presence of calcium. Gene expression, and immunostaining, revealed a relationship between MHC class II maturation and cellular plasticity. This study elucidated the role of controlled calcium supplementation under various conditions. These findings underscore the molecular mechanism of calcium-mediated immune induction and its favourable use in different calcium-containing biomaterials., essential for tissue regeneration.

Macrophage Polarization Profiling on Native and Regenerated Silk Biomaterials.

We investigated the plasticity and polarization of THP-1 cells on native and regenerated silk-based biomaterials to address the basic paradigm of immune response. Here, we report redox kinetics, adhesion morphology, and nitric oxide release patterns to identify specific subtypes of macrophages at different time points. Water-annealed silk film and native fibrous braids from Bombyx mori silkworms showed higher anti-inflammatory cytokine profiles or M2 subtypes (as evidenced by the enhanced expression of interleukin-10, interleukin-13, and interleukin-4). Ethanol-treated Bombyx mori silk films and Antheraea mylitta braids exhibited higher levels of pro-inflammatory cytokines or the M1 subtype (as evidenced by enhanced expression of interleukin-1, interleukin-6, interleukin-8, interferon-γ, TNF-α, and GM-CSF) in contact with healthy THP monocytes for 14 days; such a long study is unprecedented. Cytokine microarray analysis revealed the transition (M0-M1, M1-M2), plasticity, and stable phenotype of THP-1 cells in a variable stage in contact with different physicochemical properties of silk-based biomaterials. The detailed immunogenicity in the context of the physicochemical properties of native and regenerative silk-based biomaterials will enable us to accurately predict the possibility of a pro-/anti-inflammatory response. It will helps to predict the in vivo reprogramming and avoid fibrosis formation to enhance their clinical translational potential.

Advances in Intracellular Calcium Signaling Reveal Untapped Targets for Cancer Therapy.

Intracellular Ca2+ distribution is a tightly regulated process. Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.

Rheology and direct write printing of chitosan - graphene oxide nanocomposite hydrogels for differentiation of neuroblastoma cells.

The direct write printing method has gained popularity in synthesizing scaffolds for tissue engineering. To achieve an excellent printability of scaffolds, a thorough evaluation of rheological properties is required. We report the synthesis, characterization, rheology, and direct-write printing of chitosan - graphene oxide (CH - GO) nanocomposite hydrogels at a varying concentration of GO in 3 and 4 wt% CH polymeric gels. Rheological characterization of CH - GO hydrogels shows that an addition of only 0.5 wt% of GO leads to a substantial increase in storage modulus (G'), viscosity, and yield stress of 3 and 4 wt% of CH hydrogels. A three-interval thixotropy test (3ITT) shows that 3 wt% CH with 0.5 wt% GO hydrogel has 94% recovery of G' after 7 sequential stress cycles and is the best candidate for direct-write printing. Neuronal cell culture on 3 wt% CH with 0.5 wt% hydrogels reveals that GO promotes the differentiation of SH-SY5Y cells.

Upgrading Hepatic Differentiation and Functions on 3D Printed Silk-Decellularized Liver Hybrid Scaffolds.

We developed hybrid liver-specific three-dimensional (3D) printed scaffolds using a solubilized native decellularized liver (DCL) matrix and silk fibroin (SF) and investigated their ability to support functional cultures of hepatic cells. Rat livers were decellularized by perfusing detergents via the portal vein, solubilized using pepsin to form DCL, and characterized. SF blended with gelatin (8% w/v) was optimized with varying percentages of DCL to obtain silk gelatin-DCL bioink (SG-DCL). Different compositions of SG-DCL were studied by rheology for optimum versatility and print fidelity. 3D printed six-layered scaffolds were fabricated using a sophisticated direct-write 3D bioprinter. Huh7 cells were cultured on the 3D printed scaffolds for 3 weeks. 3D printed SG scaffolds without DCL along with 2D films (SG and SG-DCL) and 2D culture on tissue culture Petri dish control were used for comparative studies. The DCL matrix showed the absence of cells in histology and SEM. The combined SG-DCL ink at all of the studied DCL percentages (1-10%) revealed shear-thinning behavior in the printable range. The storage modulus value for the SG-DCL ink at all DCL percentages was higher than the loss modulus. In comparison to 2D controls, hepatic cells cultured on 3D SG-DCL revealed increased proliferation until 2 weeks and an upregulated expression of hepatocyte markers, including asialoglycoprotein receptor 1 (ASGR1). The Wnt pathway gene ß-catenin was upregulated by more than 4-fold in 3D SG-DCL on day 3, while it showed a decline on day 7 as compared to 3D SG and also 2D controls. The expression of the epithelial cell adhesion molecule (EpCAM) was however lower in both 2D SG-DCL (2-fold) and 3D SG-DCL (2.5-fold) as compared to that in 2D controls. Immunofluorescence studies validated the protein expression of ASGR1 in 3D SG-DCL. Albumin (ALB) was not identified on SG scaffolds but prominently expressed in 3D SG-DCL constructs. In comparison to 2D SG, both ALB (1.8-fold) and urea (5-fold) were enhanced in cells cultured on 3D SG-DCL on day 7 of culture. Hence, the SG-DCL 3D printed scaffolds provide a conducive microenvironment for elevating differentiation and functions of hepatic cells possibly through an involvement of the Wnt/ß-catenin signaling pathway.

Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting.

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure's fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs' fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.

Silk Protein Paper with In Situ Synthesized Silver Nanoparticles.

Silver nanoparticles (AgNPs) are in situ synthesized for the first time on microfibrillated silk (MFS) exfoliated from domesticated Philosamia cynthia ricini (eri) and Bombyx mori (mulberry) silkworm silk fibers. The process is rapid (hours time), does not rely on harmful chemicals, and produces robust and flexible AgNPs coated MFS (MFS-AgNPs) protein papers with excellent handling properties. None of these can be achieved by approaches used in the past to fabricate AgNPs silk systems. MFS bonds the AgNPs strongly, providing good support and stabilization for the NPs, leading to strong wash fastness. The mechanical properties of the MFS-AgNPs papers largely do not change compared to the MFS papers without nanoparticles, except for some higher concentration of AgNPs in the case of mulberry silk. The improved tensile properties of eri silk papers with or without AgNPs compared to mulberry silk papers can be attributed to the higher degree of fibrillation achieved in eri silk and its inherent higher ductility. MFS-AgNPs from eri silk also exhibit strong antibacterial activity. This study provides the basis for the development of smart protein papers based on silk fiber and functional nanomaterials.

From Orai to E-Cadherin: Subversion of Calcium Trafficking in Cancer to Drive Proliferation, Anoikis-Resistance, and Metastasis.

The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy.

Machine learning enables detection of early-stage colorectal cancer by whole-genome sequencing of plasma cell-free DNA.

BACKGROUND: Blood-based methods using cell-free DNA (cfDNA) are under development as an alternative to existing screening tests. However, early-stage detection of cancer using tumor-derived cfDNA has proven challenging because of the small proportion of cfDNA derived from tumor tissue in early-stage disease. A machine learning approach to discover signatures in cfDNA, potentially reflective of both tumor and non-tumor contributions, may represent a promising direction for the early detection of cancer. METHODS: Whole-genome sequencing was performed on cfDNA extracted from plasma samples (N = 546 colorectal cancer and 271 non-cancer controls). Reads aligning to protein-coding gene bodies were extracted, and read counts were normalized. cfDNA tumor fraction was estimated using IchorCNA. Machine learning models were trained using k-fold cross-validation and confounder-based cross-validations to assess generalization performance. RESULTS: In a colorectal cancer cohort heavily weighted towards early-stage cancer (80% stage I/II), we achieved a mean AUC of 0.92 (95% CI 0.91-0.93) with a mean sensitivity of 85% (95% CI 83-86%) at 85% specificity. Sensitivity generally increased with tumor stage and increasing tumor fraction. Stratification by age, sequencing batch, and institution demonstrated the impact of these confounders and provided a more accurate assessment of generalization performance. CONCLUSIONS: A machine learning approach using cfDNA achieved high sensitivity and specificity in a large, predominantly early-stage, colorectal cancer cohort. The possibility of systematic technical and institution-specific biases warrants similar confounder analyses in other studies. Prospective validation of this machine learning method and evaluation of a multi-analyte approach are underway.

Heterozygosity mapping for human dominant trait variants.

Homozygosity mapping is a well-known technique to identify runs of homozygous variants that are likely to harbor genes responsible for autosomal recessive disease, but a comparable method for autosomal dominant traits has been lacking. We developed an approach to map dominant disease genes based on heterozygosity frequencies of sequence variants in the immediate vicinity of a dominant trait. We demonstrate through theoretical analysis that DNA variants surrounding an inherited dominant disease variant tend to have increased heterozygosity compared with variants elsewhere in the genome. We confirm existence of this phenomenon in sequence data with known dominant pathogenic variants obtained on family members and in unrelated population controls. A computer-based approach to estimating empirical significance levels associated with our test statistics shows genome-wide p-values smaller than 0.05 for many but not all of the individuals carrying a pathogenic variant.

Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia.

PURPOSE: CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it's relevance as a therapeutic target in CLL remains understudied. EXPERIMENTAL DESIGN: We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT. RESULTS: In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. CONCLUSIONS: Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.

Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells.

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as ß-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

Silk fibroin-bioactive glass based advanced biomaterials: towards patient-specific bone grafts.

A major challenge in bone tissue engineering is to develop patient-specific, defect-site specific grafts capable of triggering specific cell signaling pathways. We could programmably fabricate the 3D printed bone constructs via direct ink writing of silk-gelatin-bioactive glass (SF-G-BG) hybrids using two different compositions of melt-derived bioactive glasses (with and without strontium) and compared against commercial 45S5 Bioglass®. Physico-chemical characterization revealed that released ions from bioactive glasses inhibited the conformational change of Bombyx mori silk fibroin protein (from random coil to ß-sheet conformation), affecting printability of the SF-G-BG ink. In-depth molecular investigations showed that strontium containing SF-G-BG constructs demonstrated superior osteogenic differentiation of mesenchymal stem cells (TVA-BMSCs) over 21 days towards osteoblastic (marked by upregulated expression of runt related transcription factor, alkaline phosphatase, osteopontin, osteonectin, integrin bone sialoprotein, osteocalcin) and osteocytic (marked by podoplanin, dentin matrix acidic phosphoprotein, sclerostin) phenotype compared to other BG compositions and silk-gelatin alone. Moreover, ionic release from bioactive glasses in the silk-gelatin ink triggered the activation of signaling pathways (BMP-2, BMP-4 and IHH), which are critical in regulating bone formation in vivo. Overall, the presence of strontium containing bioactive glass in silk-gelatin matrices provided appropriate cues in regulating the development of custom-made 3D in vitro human bone constructs.

CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A.

The Chloride Channel Accessory (CLCA) protein family was first characterized as regulators of calcium-activated chloride channel (CaCC) currents (ICaCC), but the mechanism has not been fully established. We hypothesized that CLCAs might regulate ICaCC by modulating intracellular calcium levels. In cells stably expressing human CLCA2 or vector, we found by calcium imaging that CLCA2 moderately enhanced intracellular-store release but dramatically increased store-operated entry of calcium upon cytosolic depletion. Moreover, another family member, CLCA1, produced similar effects on intracellular calcium mobilization. Co-immunoprecipitation revealed that CLCA2 interacted with the plasma membrane store-operated calcium channel ORAI-1 and the ER calcium sensor STIM-1. The effect of CLCA2 on ICaCC was tested in HEK293 stably expressing calcium-activated chloride channel TMEM16A. Co-expression of CLCA2 nearly doubled ICaCC in response to a calcium ionophore. These results unveil a new mechanism by which CLCA family members activate ICaCC and suggest a broader role in calcium-dependent processes.

Silk-Based Bioinks for 3D Bioprinting.

3D bioprinting field is making remarkable progress; however, the development of critical sized engineered tissue construct is still a farfetched goal. Silk fibroin offers a promising choice for bioink material. Nature has imparted several unique structural features in silk protein to ensure spinnability by silkworms or spider. Researchers have modified the structure-property relationship by reverse engineering to further improve shear thinning behavior, high printability, cytocompatible gelation, and high structural fidelity. In this review, it is attempted to summarize the recent advancements made in the field of 3D bioprinting in context of two major sources of silk fibroin: silkworm silk and spider silk (native and recombinant). The challenges faced by current approaches in processing silk bioinks, cellular signaling pathways modulated by silk chemistry and secondary conformations, gaps in knowledge, and future directions acquired for pushing the field further toward clinic are further elaborated.

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.