TGF-ß signaling regulates differentiation of MSCs in bone metabolism: disputes among viewpoints.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into cells of different lineages to form mesenchymal tissues, which are promising in regard to treatment for bone diseases. Their osteogenic differentiation is under the tight regulation of intrinsic and extrinsic factors. Transforming growth factor ß (TGF-ß) is an essential growth factor in bone metabolism, which regulates the differentiation of MSCs. However, published studies differ in their views on whether TGF-ß signaling regulates the osteogenic differentiation of MSCs positively or negatively. The controversial results have not been summarized systematically and the related explanations are required. Therefore, we reviewed the basics of TGF-ß signaling and summarized how each of three isoforms regulates osteogenic differentiation. Three isoforms of TGF-ß (TGF-ß1/ß2/ß3) play distinct roles in regulating osteogenic differentiation of MSCs. Additionally, other possible sources of conflicts are summarized here. Further understanding of TGF-ß signaling regulation in MSCs may lead to new applications to promote bone regeneration and improve therapies for bone diseases.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Functionally graded hydrogels with opposing biochemical cues for osteochondral tissue engineering.

Osteochondral tissue (OC) repair remains a significant challenge in the field of musculoskeletal tissue engineering. OC tissue displays a gradient structure characterized by variations in both cell types and extracellular matrix components, from cartilage to the subchondral bone. These functional gradients observed in the native tissue have been replicated to engineer OC tissuein vitro. While diverse fabrication methods have been employed to create these microenvironments, emulating the natural gradients and effective regeneration of the tissue continues to present a significant challenge. In this study, we present the design and development of CMC-silk interpenetrating (IPN) hydrogel with opposing dual biochemical gradients similar to native tissue with the aim to regenerate the complete OC unit. The gradients of biochemical cues were generated using an in-house-built extrusion system. Firstly, we fabricated a hydrogel that exhibits a smooth transition of sulfated carboxymethyl cellulose (sCMC) and TGF-ß1 (SCT gradient hydrogel) from the upper to the lower region of the IPN hydrogel to regenerate the cartilage layer. Secondly, a hydrogel with a hydroxyapatite (HAp) gradient (HAp gradient hydrogel) from the lower to the upper region was fabricated to facilitate the regeneration of the subchondral bone layer. Subsequently, we developed a dual biochemical gradient hydrogel with a smooth transition of sCMC + TGF-ß1 and HAp gradients in opposing directions, along with a blend of both biochemical cues in the middle. The results showed that the dual biochemical gradient hydrogels with biochemical cues corresponding to the three zones (i.e. cartilage, interface and bone) of the OC tissue led to differentiation of bone-marrow-derived mesenchymal stem cells to zone-specific lineages, thereby demonstrating their efficacy in directing the fate of progenitor cells. In summary, our study provided a simple and innovative method for incorporating gradients of biochemical cues into hydrogels. The gradients of biochemical cues spatially guided the differentiation of stem cells and facilitated tissue growth, which would eventually lead to the regeneration of the entire OC tissue with a smooth transition from cartilage (soft) to bone (hard) tissues. This promising approach is translatable and has the potential to generate numerous biochemical and biophysical gradients for regeneration of other interface tissues, such as tendon-to-muscle and ligament-to-bone.

Osteopontin Rejuvenates Senescent Adipose-Derived Stem Cells and Restores their Bone Tissue Regenerative Function.

The regenerative function of stem cells is compromised when the proportion of senescent stem cells increases with ageing advance. Therefore, combating stem cell senescence is of great importance for stem cell-based tissue engineering in the elderly, but remains largely unexplored. Osteopontin (OPN), a glycosylated phosphoprotein, is one of the key extracellular matrix molecules in bone tissue. OPN activates various signalling pathways and modulates cellular activities, including cell senescence. However, the role of OPN in stem cell senescence remains largely unknown. This study aims to investigate if OPN modulates cell senescence and bone regenerative function in human adipose-derived mesenchymal stem cells (ASCs), and to determine the underlying mechanisms. We first developed a senescent ASC model using serial passaging until passage 10 (P10), in which senescent cells were characterised by reduced proliferation and osteogenic differentiation capacity compared to P4 ASCs. The conditioned medium from P10 ASCs exhibited a diminished trophic effect on human osteoblasts (HOBs), compared to that from P4 ASCs. P10 ASCs on OPN-coated surface showed rejuvenated phenotype and enhanced osteogenic differentiation. The conditioned medium from P10 ASCs on OPN-coating improved trophic effects on HOBs. OPN regulated the morphology of senescent ASCs, transforming them from a more rounded and flattened cell shape to an elongated shape with a smaller area. These findings demonstrated the effects of OPN in restoring senescent ASCs functions, possibly through a mechanism that involves the modulation of cell morphology, indicating that OPN might hold a great potential for rejuvenating senescent stem cells and could potentially open a new venue for regenerating bone tissue in age-related diseases.

Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Osteocytes contribute via nuclear receptor PPAR-alpha to maintenance of bone and systemic energy metabolism.

Introduction: The view that bone and energy metabolism are integrated by common regulatory mechanisms is broadly accepted and supported by multiple strands of evidence. This includes the well-characterized role of the PPARγ nuclear receptor, which is a common denominator in energy metabolism and bone metabolism. Little is known, however, about the role of PPARα nuclear receptor, a major regulator of lipid metabolism in other organs, in bone. Methods: A side-by-side comparative study of 5-15 mo old mice with global PPARα deficiency (αKO) and mice with osteocyte-specific PPARα deficiency (αOTKO) in order to parse out the various activities of PPARα in the skeleton that are of local and systemic significance. This study included transcriptome analysis of PPARα-deficient osteocytes, and analyses of bone mass and bone microarchitecture, systemic energy metabolism with indirect calorimetry, and differentiation potential of hematopoietic and mesenchymal bone cell progenitors. These analyses were paired with in vitro studies of either intact or silenced for PPARα MLO-A5 cells to determine PPARα role in osteocyte bioenergetics. Results: In osteocytes, PPARα controls large number of transcripts coding for signaling and secreted proteins which may regulate bone microenvironment and peripheral fat metabolism. In addition, PPARα in osteocytes controls their bioenergetics and mitochondrial response to stress, which constitutes up to 40% of total PPARα contribution to the global energy metabolism. Similarly to αKO mice, the metabolic phenotype of αOTKO mice (both males and females) is age-dependent. In younger mice, osteocyte metabolism contributes positively to global energetics, however, with aging the high-energy phenotype reverts to a low-energy phenotype and obesity develops, suggesting a longitudinal negative effect of impaired lipid metabolism and mitochondrial dysfunction in osteocytes deficient in PPARα. However, bone phenotype was not affected in αOTKO mice except in the form of an increased volume of marrow adipose tissue in males. In contrast, global PPARα deficiency in αKO mice led to enlarged bone diameter with a proportional increase in number of trabeculae and enlarged marrow cavities; it also altered differentiation of hematopoietic and mesenchymal marrow cells toward osteoclast, osteoblast and adipocyte lineages, respectively. Discussion: PPARα role in bone is multileveled and complex. In osteocytes, PPARα controls the bioenergetics of these cells, which significantly contributes to systemic energy metabolism and their endocrine/paracrine function in controlling marrow adiposity and peripheral fat metabolism.

Mast4 determines the cell fate of MSCs for bone and cartilage development.

Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-ß and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-ß1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3ß and subsequent Smurf1 recruitment, promoted ß-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4-/- mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

Klotho in Osx+-mesenchymal progenitors exerts pro-osteogenic and anti-inflammatory effects during mandibular alveolar bone formation and repair.

Maxillofacial bone defects are commonly seen in clinical practice. A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration. The fibroblast growth factor (FGF) signalling pathway is critical for the development of maxillofacial bone. Klotho, a type I transmembrane protein, is an important components of FGF receptor complexes. Recent studies have reported the presence of Klotho expression in bone. However, the role of Klotho in cranioskeletal development and repair remains unknown. Here, we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions. Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo. Under conditions of inflammation and trauma-induced bone loss, we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression. More importantly, we show for the first time that Klotho is present in human alveolar bone, with a distinct expression pattern under both normal and pathological conditions. In summary, our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair. Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration. It may also be a target for future therapeutics.

Age-Progressive and Gender-Dependent Bone Phenotype in Mice Lacking Both Ebf1 and Ebf2 in Prrx1-Expressing Mesenchymal Cells.

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.

Mesenchymal stromal cell-derived septoclasts resorb cartilage during developmental ossification and fracture healing.

Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.

Supercritical carbon dioxide dried double layer laponite XLS and alginate/polyacrylamide construct and immune response.

Fabrication of immunocompatible tissue constructs for bone-cartilage defect regeneration is of prime importance. In this study, a double layer hydrogel was successfully synthesized, where alginate/polyacrylamide were formulated to represent cartilage layer (5-10 % (w/w) total polymer ratio) and laponite XLS (2-5-8% (w/w))/alginate/polyacrylamide formed bone layer. Hydrogels were dried by supercritical CO2 at 100 and 200 bar, 45 °C, 5 g/min CO2 flow rate for 2 h. Constructs were treated with collagen, then cellularized and embedded in cell-laden GelMA to mimic the cellular microenvironment. The optimum weight ratio of alginate/polyacrylamide:laponite XLS was 10:5 based on mechanical strength test results. The constructs yielded high porosity (91.50 m2/g) and mesoporous structure, owing to the diffusivity of CO2 at 200 bar (0.49 × 10-7 m2/s). Constructs were then treated with collagen to increase cell adhesion and ATDC5 cells were seeded in the cartilage layer, whereas hFOB cells to the bone layer. About 10-15 % higher cell viability was attained. The porous structure of the construct allowed infiltration of macrophages, promoted polarization and positively affected the behavior of macrophages, yielding a decrease in M1 markers, whereas an increase in M2 on day 4. The formulated tissue constructs would be of value in tissue engineering applications.

Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools.

Leptin receptor (LepR)-positive cells are key components of the bone marrow hematopoietic microenvironment, and highly enrich skeletal stem and progenitor cells that maintain homeostasis of the adult skeleton. However, the heterogeneity and lineage hierarchy within this population has been elusive. Using genetic lineage tracing and single-cell RNA sequencing, we found that Lepr-Cre labels most bone marrow stromal cells and osteogenic lineage cells in adult long bones. Integrated analysis of Lepr-Cre-traced cells under homeostatic and stress conditions revealed dynamic changes of the adipogenic, osteogenic, and periosteal lineages. Importantly, we discovered a Notch3+ bone marrow sub-population that is slow-cycling and closely associated with the vasculatures, as well as key transcriptional networks promoting osteo-chondrogenic differentiation. We also identified a Sca-1+ periosteal sub-population with high clonogenic activity but limited osteo-chondrogenic potential. Together, we mapped the transcriptomic landscape of adult LepR+ stem and progenitor cells and uncovered cellular and molecular mechanisms underlying their maintenance and lineage specification.

Role of Mel1/Prdm16 in bone differentiation and morphology.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16-/-) was embryonic lethal at E 11.5. Heterozygous mice showed abnormal cartilage and bone formation in the postnatal skull and long bones, suggesting that Mel1/Prdm16 expression plays an important role in bone development. In osteoblast and chondrocyte cell lines, Mel1/Prdm16 promotes the differentiation of chondrocytes and regulates the differentiation of osteoblasts. Transient repression of the master regulator Runx2 is required for chondrocyte differentiation at an early stage of differentiation. However, in Mel1/Prdm16-suppressed ATDC5 cells, the initial suppression of Runx2 was lacking and its expression was upregulated at the beginning of differentiation, suggesting that chondrogenic differentiation is suppressed in Mel1/Prdm16+/- mesenchymal progenitor cells because Runx2 expression is upregulated during the early stage of differentiation. Thus, the Mel1/Prdm16 gene may be involved in the early repression of Runx2 expression during osteochondral differentiation and promote chondrogenic differentiation.

An In Vitro Evaluation of the Biological and Osteogenic Properties of Magnesium-Doped Bioactive Glasses for Application in Bone Tissue Engineering.

Magnesium (Mg2+) is known to play a crucial role in mineral and matrix metabolism of bone tissue and is thus increasingly considered in the field of bone tissue engineering. Bioactive glasses (BGs) offer the promising possibility of the incorporation and local delivery of therapeutically active ions as Mg2+. In this study, two Mg2+-doped derivatives of the ICIE16-BG composition (49.46 SiO2, 36.27 CaO, 6.6 Na2O, 1.07 P2O5, 6.6 K2O (mol%)), namely 6Mg-BG (49.46 SiO2, 30.27 CaO, 6.6 Na2O, 1.07 P2O5, 6.6 K2O, 6.0 MgO (mol%) and 3Mg-BG (49.46 SiO2, 33.27 CaO, 6.6 Na2O, 1.07 P2O5, 6.6 K2O, 3.0 MgO (mol%)) were examined. Their influence on viability, proliferation and osteogenic differentiation of human mesenchymal stromal cells (MSCs) was explored in comparison to the original ICIE16-BG. All BGs showed good biocompatibility. The Mg2+-doped BGs had a positive influence on MSC viability alongside with inhibiting effects on MSC proliferation. A strong induction of osteogenic differentiation markers was observed, with the Mg2+-doped BGs significantly outperforming the ICIE16-BG regarding the expression of genes encoding for protein members of the osseous extracellular matrix (ECM) at certain observation time points. However, an overall Mg2+-induced enhancement of the expression of genes encoding for ECM proteins could not be observed, possibly due to a too moderate Mg2+ release. By adaption of the Mg2+ release from BGs, an even stronger impact on the expression of genes encoding for ECM proteins might be achieved. Furthermore, other BG-types such as mesoporous BGs might provide a higher local presence of the therapeutically active ions and should therefore be considered for upcoming studies.

Human Periodontal Ligament Stem Cell and Umbilical Vein Endothelial Cell Co-Culture to Prevascularize Scaffolds for Angiogenic and Osteogenic Tissue Engineering.

(1) Background: Vascularization remains a critical challenge in bone tissue engineering. The objective of this study was to prevascularize calcium phosphate cement (CPC) scaffold by co-culturing human periodontal ligament stem cells (hPDLSCs) and human umbilical vein endothelial cells (hUVECs) for the first time; (2) Methods: hPDLSCs and/or hUVECs were seeded on CPC scaffolds. Three groups were tested: (i) hUVEC group (hUVECs on CPC); (ii) hPDLSC group (hPDLSCs on CPC); (iii) co-culture group (hPDLSCs + hUVECs on CPC). Osteogenic differentiation, bone mineral synthesis, and microcapillary-like structures were evaluated; (3) Results: Angiogenic gene expressions of co-culture group were 6-9 fold those of monoculture. vWF expression of co-culture group was 3 times lower than hUVEC-monoculture group. Osteogenic expressions of co-culture group were 2-3 folds those of the hPDLSC-monoculture group. ALP activity and bone mineral synthesis of co-culture were much higher than hPDLSC-monoculture group. Co-culture group formed capillary-like structures at 14-21 days. Vessel length and junction numbers increased with time; (4) Conclusions: The hUVECs + hPDLSCs co-culture on CPC scaffold achieved excellent osteogenic and angiogenic capability in vitro for the first time, generating prevascularized networks. The hPDLSCs + hUVECs co-culture had much better osteogenesis and angiogenesis than monoculture. CPC scaffolds prevacularized via hPDLSCs + hUVECs are promising for dental, craniofacial, and orthopedic applications.

Diagnosis of bone metastases in breast cancer: Lesion-based sensitivity of dual-time-point FDG-PET/CT compared to low-dose CT and bone scintigraphy.

We compared lesion-based sensitivity of dual-time-point FDG-PET/CT, bone scintigraphy (BS), and low-dose CT (LDCT) for detection of various types of bone metastases in patients with metastatic breast cancer. Prospectively, we included 18 patients with recurrent breast cancer who underwent dual-time-point FDG-PET/CT with LDCT and BS within a median time interval of three days. A total of 488 bone lesions were detected on any of the modalities and were categorized by the LDCT into osteolytic, osteosclerotic, mixed morphologic, and CT-negative lesions. Lesion-based sensitivity was 98.2% (95.4-99.3) and 98.8% (96.8-99.5) for early and delayed FDG-PET/CT, respectively, compared with 79.9% (51.1-93.8) for LDCT, 76.0% (36.3-94.6) for BS, and 98.6% (95.4-99.6) for the combined BS+LDCT. BS detected only 51.2% of osteolytic lesions which was significantly lower than other metastatic types. SUVs were significantly higher for all lesion types on delayed scans than on early scans (P<0.0001). Osteolytic and mixed-type lesions had higher SUVs than osteosclerotic and CT-negative metastases at both time-points. FDG-PET/CT had significantly higher lesion-based sensitivity than LDCT and BS, while a combination of the two yielded sensitivity comparable to that of FDG-PET/CT. Therefore, FDG-PET/CT could be considered as a sensitive one-stop-shop in case of clinical suspicion of bone metastases in breast cancer patients.

Down-regulation of VCAM-1 in bone mesenchymal stem cells reduces inflammatory responses and apoptosis to improve cardiac function in rat with myocardial infarction.

BACKGROUND: Bone mesenchymal stem cells (BMSCs) has been well known to exert therapeutic potential for patients with myocardial infarction (MI). VCAM-1 can promote the migration of lymphocytes to the inflammatory zone. In the present study, we tried to explore whether VCAM-1 silenced-BMSCs have better therapeutic effects on MI. METHODS: BMSCs were isolated and cultured followed by treatment of a lentivirus silencing VCAM-1 and NF-κB activator (PMA). Besides, MI rat models were also established and injected with treated BMSCs to detect the effect of VCAM-1 silenced-BMSCs in MI, as evidenced by detection of cardiac function, survival of rats within 72 h, infarct size and myocardial cell apoptosis. Moreover, the expression of NF-κB-regulated gene products was also determined. RESULTS: The implantation of sh-VCAM-1 BMSCs into MI rats resulted in more reductions in myocardial infarct size as well as myocardial cell apoptosis, improved cardiac function, the number of survived rats within 72 h, and survival time within 72 h compared with the individual treatments of either BMSCs or control. In addition, transplanted BMSCs down-regulated the expression of NF-κB-p65, MMP-9, TNF-α, and Bax, and up-regulated VEGF and Bcl-2 in myocardial tissue, which could be further enhanced by sh-VCAM-1 and rescued by PMA. CONCLUSION: Our study demonstrated that silencing VCAM-1 in BMSCs could inhibit inflammation and apoptosis, thus improving cardiac function in MI.

Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option.

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds.

Bioresorbable biomaterials can fill bone defects and act as temporary scaffold to recruit MSCs to stimulate their differentiation. Among the different bioresorbable polymers studied, this work focuses on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL). Were prepared blends of PHBV and PCL to obtain PHBV based biomaterials with good tenacity, important for bone tissue repair, associated with biocompatible properties of PCL. This study assesses the viability of Vero cells on scaffolds of PHBV, PCL, and their blends and the osteogenic differentiation of mesenchymal stem cells (MSCs). Materials were characterized in surface morphology, DSC and Impact Strength (IS). Vero cells and MSCs were assessed by MTT assay, cytochemical and SEM analysis. MSC osteogenic differentiation was evaluated through alizarin red staining and ALP activity. We found some roughness onto surface materials. DSC showed that the blends presented two distinct melting peaks, characteristic of immiscible blends. IS test confirmed that PHBV-PCL blends is an alternative for increase the tenacity of PHBV. MTT assay showed cells with high metabolic activities on extract toxicity test, but with low activity in the direct contact test. SEM analysis showed spreading cells with irregular and flattened morphology on different substrates. Cytochemical study revealed that MSCs maintained their morphology, although in smaller number for MSCs. The development of nodules of mineralized organic matrix in MSC cultures was identified by alizarin red staining and osteogenic differentiation was confirmed by the quantification of ALP activity. Thus, our scaffolds did not interfere on viability of Vero cells or the osteogenic differentiation of MSCs.

Short-Term Evaluation of Cellular Fate in an Ovine Bone Formation Model.

The ovine critical-sized defect model provides a robust preclinical model for testing tissue-engineered constructs for use in the treatment of non-union bone fractures and severe trauma. A critical question in cell-based therapies is understanding the optimal therapeutic cell dose. Key to defining the dose and ensuring successful outcomes is understanding the fate of implanted cells, e.g., viability, bio-distribution and exogenous infiltration post-implantation. This study evaluates such parameters in an ovine critical-sized defect model 2 and 7 days post-implantation. The fate of cell dose and behaviour post-implantation when combined with nanomedicine approaches for multi-model tracking and remote control using external magnetic fields is also addressed. Autologous STRO-4 selected mesenchymal stromal cells (MSCs) were labelled with a fluorescent lipophilic dye (CM-Dil), functionalised magnetic nanoparticles (MNPs) and delivered to the site within a naturally derived bone extracellular matrix (ECM) gel. Encapsulated cells were implanted within a critical-sized defect in an ovine medial femoral condyle and exposed to dynamic gradients of external magnetic fields for 1 h per day. Sheep were sacrificed at 2 and 7 days post-initial surgery where ECM was harvested. STRO-4-positive (STRO-4+) stromal cells expressed osteocalcin and survived within the harvested gels at day 2 and day 7 with a 50% loss at day 2 and a further 45% loss at 7 days. CD45-positive leucocytes were also observed in addition to endogenous stromal cells. No elevation in serum C-reactive protein (CRP) or non-haem iron levels was observed following implantation in groups containing MNPs with or without magnetic field gradients. The current study demonstrates how numbers of therapeutic cells reduce substantially after implantation in the repair site. Cell death is accompanied by enhanced leucocyte invasion, but not by inflammatory blood marker levels. Crucially, a proportion of implanted STRO-4+ stromal cells expressed osteocalcin, which is indicative of osteogenic differentiation. Furthermore, MNP labelling did not alter cell number or result in a further deleterious impact on stromal cells following implantation.