Yun Wang

The synergistic effects of 3-D porous silk fibroin matrix scaffold properties and hydrodynamic environment in cartilage tissue regeneration

Autologous cell-based tissue engineering using three-dimensional porous scaffolds has provided a good option for the repair of cartilage defects. Silk fibroin-based scaffolds are naturally degradable materials with excellent biocompatibility and robust mechanical properties, indicating potential applications in cartilage tissue engineering. In this study, silk fibroin scaffolds prepared by freeze-drying (FD) and salt-leaching (SL300 and SL500) were fully characterized and used to study the effects of silk fibroin scaffold properties on chondrocyte attachment, proliferation and differentiation. The synergistic effects of scaffold properties and hydrodynamic environment generated by in vitro rocking culture were also investigated using static cultures as control. FD scaffolds with small pore size and lower porosity increased cell attachment but inhibited cell penetration and limited cell proliferation and differentiation. In contrast, SL scaffolds displaying a bigger pore size, higher porosity and crystallinity resulted in homogenous cell distribution, increasing cell proliferation and advanced chondrocyte differentiation in terms of their spherical morphology, predominant chondrogenic gene expression and abundant cartilaginous extracellular matrix production. A hydrodynamic environment was beneficial to chondrocyte proliferation, differentiation, and integrin gene expression in a pore size dependent manner with superior cartilage matrix production but limited hypertrophic differentiation obtained using chondrocyte-seeded SL500 scaffolds. Integrin alpha5beta1 might mediate these effects. Chondrocyte/SL500 silk fibroin constructs obtained under in vitro rocking culture might serve as an excellent implant for in vivo cartilage defect reparation.

Protein-disulfide Isomerase-associated 3 (Pdia3) Mediates the Membrane Response to 1,25-Dihydroxyvitamin D3 in Osteoblasts

Protein-disulfide isomerase-associated 3 (Pdia3) is a multifunctional protein hypothesized to be a membrane receptor for 1,25(OH)2D3. In intestinal epithelium and chondrocytes, 1,25(OH)2D3 stimulates rapid membrane responses that are different from genomic effects via the vitamin D receptor (VDR). In this study, we show that 1,25(OH)2D3 stimulates phospholipase A2 (PLA2)-dependent rapid release of prostaglandin E2 (PGE2), activation of protein kinase C (PKC), and regulation of bone-related gene transcription and mineralization in osteoblast-like MC3T3-E1 cells (WT) via a mechanism involving Pdia3. Pdia3 was present in caveolae based on co-localization with lipid rafts and caveolin-1. In Pdia3-silenced (Sh-Pdia3) cells, 1,25(OH)2D3 failed to stimulate PKC and PGE2 responses; in Pdia3-overexpressing cells (Ov-Pdia3), responses to 1,25(OH)2D3 were augmented. Downstream mediators of Pdia3, PLA2-activating protein (PLAA) and arachidonic acid, stimulated similar PKC activation in wild-type, Sh-Pdia3, and Ov-Pdia3 cells supporting the hypothesis that Pdia3 mediates the membrane action of 1,25(OH)2D3. Treatment of MC3T3-E1 cells with 1,25(OH)2D3 for 9 min stimulated rapid phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased expression of alkaline phosphatase, MMP-13, and osteopontin but decreased expression of osteocalcin, osteoprotegerin (mRNA and protein), and smad2. These effects were attenuated in Sh-Pdia3 cells. Sh-Pdia3 cells produced higher numbers of von Kossa-positive nodules and alizarin red-positive nodules compared with WT cells with or without 1,25(OH)2D3 treatment whereas Ov-Pdia3 did not show any mineralization. Our data suggest Pdia3 is an important initiator of 1,25(OH)2D3-stimulated membrane signaling pathways, which have both genomic and non genomic effects during osteoblast maturation.

Engineering three-dimensional stem cell morphogenesis for the development of tissue models and scalable regenerative therapeutics.

The physiochemical stem cell microenvironment regulates the delicate balance between self-renewal and differentiation. The three-dimensional assembly of stem cells facilitates cellular interactions that promote morphogenesis, analogous to the multicellular, heterotypic tissue organization that accompanies embryogenesis. Therefore, expansion and differentiation of stem cells as multicellular aggregates provides a controlled platform for studying the biological and engineering principles underlying spatiotemporal morphogenesis and tissue patterning. Moreover, three-dimensional stem cell cultures are amenable to translational screening applications and therapies, which underscores the broad utility of scalable suspension cultures across laboratory and clinical scales. In this review, we discuss stem cell morphogenesis in the context of fundamental biophysical principles, including the three-dimensional modulation of adhesions, mechanics, and molecular transport and highlight the opportunities to employ stem cell spheroids for tissue modeling, bioprocessing, and regenerative therapies.

Disruption of Pdia3 gene results in bone abnormality and affects 1α,25-dihydroxy-vitamin D3-induced rapid activation of PKC☆

1,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3] is a critical regulator of bone development. Protein disulfide isomerase A3 (Pdia3) is a multifunctional protein that has been associated with rapid membrane-initiated signalling by 1alpha,25(OH)2D3 in several cell types. To identify the physiological roles of Pdia3 in skeletal development, we generated Pdia3-deficient mice. No homozygous mice were observed at birth, indicating that the targeted disruption of the Pdia3 gene resulted in embryonic lethality. Pdia3 deficiency also resulted in skeletal manifestations as revealed by muCT analysis of femurs from 15-week-old heterozygous mice. The Pdia3+/- mice had increased metaphyseal bone volume and trabeculae compared to Pdia3+/+ mice. In contrast, the area and thickness of cortical bone at the femoral mid-diaphysis of Pdia3+/+ mice significantly exceeded that of Pdia3+/- mice. In vitro studies in osteoblast-like MC3T3-E1 cells showed that silencing of Pdia3 abolished 1alpha,25(OH)2D3-induced rapid activation of protein kinase C (PKC) while overexpression of Pdia3 resulted in augmentation of PKC activity by 1alpha,25(OH)2D3. Taken together, these data indicated that Pdia3 plays a crucial role in 1alpha,25(OH)2D3-regulated bone formation and the Pdia3-PKC signalling pathway might be involved in this process.

Rapid membrane responses to dihydrotestosterone are sex dependent in growth plate chondrocytes

Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M DHT (p<0.05), whereas testosterone had no effect. PKC activity was augmented at 9 and 90 min, and then decreased to baseline at 270min. Neither testosterone nor DHT affected PKC in female cells. U73122, quinacrine, and AACOCF3 inhibited DHT-induced activation of PKC. DHT treatment for 9 min had no effect in [(3)H]-thymidine incorporation in quiescent confluent cultures but caused a dose dependent increase in alkaline phosphatase specific activity. Inhibition of PLC reduced the response of to DHT in a dose dependent manner, indicating that PLC is involved. In conclusion, our study indicates that DHT, but not testosterone, has sex-specific rapid membrane effects in male growth plate chondrocytes involving PLC and PLA2-mediated PKC signaling pathways. Together with previous observations showing that male cells convert testosterone to DHT, these results suggest that DHT might act in the membrane through an autocrine/paracrine mechanism.

MMP-mediated mesenchymal morphogenesis of pluripotent stem cell aggregates stimulated by gelatin methacrylate microparticle incorporation

Matrix metalloproteinases (MMPs) remodel the extracellular matrix (ECM) to facilitate epithelial-to-mesenchymal transitions (EMTs) and promote cell specification during embryonic development. In this study, we hypothesized that introducing degradable ECM-based biomaterials to pluripotent stem cell (PSC) aggregates would modulate endogenous proteolytic activity and consequently enhance the differentiation and morphogenesis within 3D PSC aggregates. Gelatin methacrylate (GMA) microparticles (MPs) of low (∼20%) or high (∼90%) cross-linking densities were incorporated into mouse embryonic stem cell (ESC) aggregates, and the effects on MMP activity and cell differentiation were examined with or without MMP inhibition. ESC aggregates containing GMA MPs expressed significantly higher levels of total MMP and MMP-2 than aggregates without MPs. GMA MP incorporation increased expression of EMT markers and enhanced mesenchymal morphogenesis of PSC aggregates. MMP inhibition completely abrogated these effects, and GMA MP-induced MMP activation within ESC aggregates was partially reduced by pSMAD 1/5/8 inhibition. These results suggest that GMA particles activate MMPs by protease-substrate interactions to promote EMT and mesenchymal morphogenesis of ESC aggregates in an MMP-dependent manner. We speculate that controlling protease activity via the introduction of ECM-based materials may offer a novel route to engineer the ECM microenvironment to modulate stem cell differentiation.

Sex-specific response of rat costochondral cartilage growth plate chondrocytes to 17β-estradiol involves differential regulation of plasma membrane associated estrogen receptors

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2-3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2s effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Osteogenic Embryoid Body-Derived Material Induces Bone Formation In Vivo

The progressive loss of endogenous regenerative capacity that accompanies mammalian aging has been attributed at least in part to alterations in the extracellular matrix (ECM) composition of adult tissues. Thus, creation of a more regenerative microenvironment, analogous to embryonic morphogenesis, may be achieved via pluripotent embryonic stem cell (ESC) differentiation and derivation of devitalized materials as an alternative to decellularized adult tissues, such as demineralized bone matrix (DBM). Transplantation of devitalized ESC materials represents a novel approach to promote functional tissue regeneration and reduce the inherent batch-to-batch variability of allograft-derived materials. In this study, the osteoinductivity of embryoid body-derived material (EBM) was compared to DBM in a standard in vivo ectopic osteoinduction assay in nude mice. EBM derived from EBs differentiated for 10 days with osteogenic media (+β-glycerophosphate) exhibited similar osteoinductivity to active DBM (osteoinduction score = 2.50 ± 0.27 vs. 2.75 ± 0.16) based on histological scoring, and exceeded inactive DBM (1.13 ± 0.13, p < 0.005). Moreover, EBM stimulated formation of new bone, ossicles, and marrow spaces, similar to active DBM. The potent osteoinductivity of EBM demonstrates that morphogenic factors expressed by ESCs undergoing osteogenic differentiation yield a novel devitalized material capable of stimulating de novo bone formation in vivo.

Mineral particles modulate osteo-chondrogenic differentiation of embryonic stem cell aggregates.

UNLABELLED Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications. STATEMENT OF SIGNIFICANCE Directing stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo. This hybrid biomaterial-ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3 +/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3 +/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3 +/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3 +/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3 +/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.