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.

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.

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.

Microarray meta-analysis identifies evolutionarily conserved BMP signaling targets in developing long bones.

In vertebrates, BMP signaling has been demonstrated to be sufficient for bone formation in several tissue contexts. This suggests that genes necessary for bone formation are expressed in a BMP signaling dependent manner. However, till date no gene has been reported to be expressed in a BMP signaling dependent manner in bone. Our aim was to identify such genes. On searching the literature we found that several microarray experiments have been conducted where the transcriptome of osteogenic cells in absence and presence of BMP signaling activation have been compared. However, till date, there is no evidence to suggest that any of the genes found to be upregulated in presence of BMP signaling in these microarray analyses is indeed a target of BMP signaling in bone. We wanted to utilize this publicly available information to identify candidate BMP signaling target genes in vivo. We performed a meta-analysis of six such comparable microarray datasets. This analysis and subsequent experiments led to the identification of five targets of BMP signaling in bone that are conserved both in mouse and chick. Of these Lox, Klf10 and Gpr97 are likely to be direct transcriptional targets of BMP signaling pathway. Dpysl3, is a novel BMP signaling target identified in our study. Our data demonstrate that Dpysl3 is important for osteogenic differentiation of mesenchymal cells and is involved in cell secretion. We have demonstrated that the expression of Dpysl3 is co-operatively regulated by BMP signaling and Runx2. Based on our experimental data, in silico analysis of the putative promoter-enhancer regions of Bmp target genes and existing literature, we hypothesize that BMP signaling collaborates with multiple signaling pathways to regulate the expression of a unique set of genes involved in endochondral ossification.

BMP signaling in development and diseases: a pharmacological perspective.

Bone morphogenetic protein (BMP) signaling has been implicated in several processes during embryonic development and in adult tissue homeostasis. Maintenance of many organs such as skin, intestinal villi, bones and bone marrow requires continuous regeneration and subsequent differentiation of stem cells in order to maintain organ shape and size necessary for proper functioning. Although BMPs were initially identified as osteogenic factors present in demineralized bone capable of inducing ectopic bone formation, it is now evident that BMPs perform several other functions during embryonic development as well as during the adult life of an organism. Many disorders have been linked to either the BMPs or the molecules functioning downstream of BMP signaling pathway. This review summarizes the existing literature describing the role of BMP signaling during embryonic development and in adult tissue homeostasis to provide a perspective on pharmacological interventions of BMP signaling pathway to mitigate several disease conditions.

BMP4 is dispensable for skeletogenesis and fracture-healing in the limb.

Bone morphogenetic proteins (BMPs) are potent bone-forming agents that show clinical efficacy when used in patients to augment fracture-healing. Molecular profiling of fracture tissues has confirmed that BMPs 2, 3, 4, 5, 6, and 7 are expressed during the healing process, and it has identified a specific temporal pattern of expression for each BMP. Mice engineered to express increased levels of BMP antagonists have fragile bones that are prone to fracture, suggesting that BMPs not only mediate bone formation in the context of repair, but may also have a role in maintaining adult bone. In this study, mice carrying floxed Bmp4 alleles were bred with Prx1-cre transgenic mice to establish limb-specific removal of Bmp4. We compared these mice to mice in which Bmp2 was specifically deleted from the limb, and we then assessed limb skeletogenesis and fracture-healing. Limb skeletogenesis occurs normally in the absence of BMP4, and postnatal skeletal growth was also unaffected when BMP4 was removed. When mice lacking BMP4 were challenged to repair fractures, they were able to mount a successful healing response. We concluded that BMP4 is not required for formation of the limb skeleton and that femur fracture-healing is unaffected by the absence of BMP4. This study demonstrates that BMP4 is not required for bone formation and function in the limb, giving us further insights into the utility of recombinant human BMPs as therapeutic agents.

The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate.

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.

Role of the Tsc1-Tsc2 complex in signaling and transport across the cell membrane in the fission yeast Schizosaccharomyces pombe.

Heterozygous inactivation of either human TSC1 or TSC2 causes tuberous sclerosis (TSC), in which development of benign tumors, hamartomas, occurs via a two-hit mechanism. In this study, fission yeast genes homologous to TSC1 and TSC2 were identified, and their protein products were shown to physically interact like the human gene products. Strains lacking tsc1(+) or tsc2(+) were defective in uptake of nutrients from the environment. An amino acid permease, which is normally positioned on the plasma membrane, aggregated in the cytoplasm or was confined in vacuole-like structures in Deltatsc1 and Deltatsc2 strains. Deletion of tsc1(+) or tsc2(+) also caused a defect in conjugation. When a limited number of the cells were mixed, they conjugated poorly. The conjugation efficiency was improved by increased cell density. Deltatsc1 cells were not responsive to a mating pheromone, P-factor, suggesting that Tsc1 has an important role in the signal cascade for conjugation. These results indicate that the fission yeast Tsc1-Tsc2 complex plays a role in the regulation of protein trafficking and suggest a similar function for the human proteins. We also show that fission yeast Int6 is involved in a similar process, but functions in an independent genetic pathway.