Effects of TGF-β3 and preculture period of osteogenic cells on the chondrogenic differentiation of rabbit marrow mesenchymal stem cells encapsulated in a bilayered hydrogel composite

In this work, injectable, biodegradable hydrogel composites of crosslinked oligo(poly(ethylene glycol) fumarate) and gelatin microparticles (MPs) were used to fabricate a bilayered osteochondral construct. Rabbit marrow mesenchymal stem cells (MSCs) were encapsulated with transforming growth factor-β3 (TGF-β3)-loaded MPs in the chondrogenic layer and cocultured with cells of different periods of osteogenic preculture (0, 3, 6 and 12 days) in the osteogenic layer to investigate the effects of TGF-β3 delivery and coculture on the proliferation and differentiation of cells in both layers. The results showed that, in the chondrogenic layer, TGF-β3 significantly stimulated chondrogenic differentiation of MSCs. In addition, cells of various osteogenic preculture periods in the osteogenic layer, along with TGF-β3, enhanced gene expression for MSC chondrogenic markers to different extents. In the osteogenic layer, cells maintained their alkaline phosphatase activity during the coculture; however, mineralization was delayed by the presence of TGF-β3. Overall, this study demonstrated the fabrication of bilayered hydrogel composites which mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers, while illustrating that encapsulated cells of different degrees of osteogenic differentiation can significantly influence the chondrogenic differentiation of cocultured progenitor cells in both the presence and absence of chondrogenic growth factors.     

TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(?-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.     

The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin–polysaccharide cardiac patches in vitro

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS–hyaluronic acid (SF/CS–HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR–FTIR spectra confirm the co-existence of CS or CS–HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS–HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P < 0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS–HA hybrid patches were significantly better (P < 0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS–HA patches were 100%, 282.9 ± 6.5%, 337.0 ± 8.0% and 332.6 ± 6.6% (n = 6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS–HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P < 0.01 or better, n = 3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS–HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS–HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS–HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.     

Investigating the effect of hypoxic culture on the endothelial differentiation of human amniotic fluid‐derived stem cells

Amniotic fluid‐derived stem cells (AFSCs) are a unique stem cell source that may have great potential for use in tissue engineering (TE) due to their pluripotentiality. AFSCs have previously shown angiogenic potential and may present an alternative cell source for endothelial‐like cells that could be used in range of applications, including the pre‐vascularisation of TE constructs and the treatment of ischaemic diseases. This study investigated the ability of these cells to differentiate down an endothelial lineage with the aim of producing an endothelial‐like cell suitable for use in pre‐vascularisation. As hypoxia and the associated HIF‐1 pathway have been implicated in the induction of angiogenesis in a number of biological processes, it was hypothesised that culture in hypoxic conditions could enhance the endothelial differentiation of AFSCs. The cells were cultured in endothelial cell media supplemented with 50 ng mL⁻¹ of VEGF, maintained in normoxia, intermittent hypoxia or continuous hypoxia and assessed for markers of endothelial differentiation at day 7 and 14. The results demonstrated that AFSCs subjected to these culture conditions display an endothelial gene expression profile and adopted functional endothelial cell characteristics indicative of early endothelial differentiation. Culture in continuous hypoxia enhanced endothelial gene expression but did not enhance functional endothelial cell characteristics. Overall, AFSCs subjected to endothelial stimuli demonstrated a less mature endothelial gene expression profile and phenotype when compared with HUVECs, the endothelial cell control. However, this study is the first time that the positive effect of an extended period of continuous hypoxic culture on endothelial differentiation in AFSCs has been demonstrated.     

Effect of temporally patterned TNF-α delivery on in vitro osteogenic differentiation of mesenchymal stem cells cultured on biodegradable polymer scaffolds

Recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), in bone regeneration has heralded a new direction in the design of tissue engineering constructs. Previous studies have demonstrated that continuous delivery of 50?ng/ml TNF-α to mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) biodegradable electrospun poly(?-caprolactone) (PCL) microfiber meshes stimulates mineralized matrix deposition, a marker of osteogenic differentiation. Since TNF-α exhibits a biphasic pattern of expression following bone fracture in vivo, this study aimed to investigate the effects of temporal patterns of TNF-α delivery on in vitro osteogenic differentiation of MSCs cultured on 3D electrospun PCL scaffolds. MSCs were cultured for 16?days and exposed to continuous, early, intermediate, or late TNF-α delivery. To further elucidate the effects of TNF-α on osteogenic differentiation, the study design included MSCs precultured both in the presence and absence of typically required osteogenic supplement dexamethasone. Mineralized matrix deposition was not observed in constructs with dexamethasone-naïve MSCs, suggesting that TNF-α is not sufficient to trigger in vitro osteogenic differentiation of MSCs. For MSCs precultured with dexamethasone, TNF-α suppressed alkaline phosphatase activity, an early marker of osteogenic differentiation, and stimulated mineralized matrix deposition, a late stage marker of MSC osteogenic differentiation. By elucidating the impact of temporal variations in TNF-α delivery on MSC osteogenic differentiation, our results offer insight into the regenerative mechanism of TNF-α and provide the design parameters for a novel tissue engineering strategy that rationally controls TNF-α signaling to stimulate bone regeneration.     

Influence of Wnt/beta-catenin signal transduction pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells北大核心

BACKGROUND: Umbilical cord mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells in vitro and in vivo. However, the exact mechanism is still unknown. [Existing studies have shown that the Wnt/β-catenin signaling pathway is closely related to this process. OBJECTIVE: To explore the effect of Wnt/β-catenin signaling pathway on the differentiation of umbilical cord mesenchymal stem cells into hepatocyte-like cells and its potential molecular mechanism. METHODS: Human umbilical cord mesenchymal stem cells were extracted from the neonatal umbilical cord by tissue adherent method. After being cultured and purified, the umbilical cord mesenchymal stem cells at passages 4-6 were divided into four groups: control group (DMEM culture group), hepatocyte-like differentiation group, activator Wnt3a group (adding 20 µg/L Wnt3a, an activator of Wnt/β-catenin signaling pathway, under the differentiation condition), ond inhibitor Dkk-1 group (adding 20 µg/L Dkk-1, on inhibitor of Wnt/β-catenin signaling pathway, under the differentiation condition). Induced cells were collected respectively on days 7,14, 21,28. Their mRNA and protem expressions of α-fetoprotein (AFP), albumin (ALB), hepatocytt nuclear factor 4α (HNF4α) and Cytokeratin-19 (CK-19) in the cells were detcted by real-time quantitative PCR and western blot respectively. (Meanwhile, Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test were applied to Oetect the function of hepatocyte-like cells. RESULTS AND CONCLUSION: Compared with the control group, expressions of AFP and HNF4a mRNA and protein as well as ALB mRNA were significantly up-regulated in the hepatocyte-like differentiation group, cctivator Wnt3a group end inhibitor Dkk-1 group (P < 0.05). Whereas, there was a decrease in the CK-19 expression at mRNA and protein levels (P < 0.01) in these three groups. Compared with the hepatocyte-like differentiation group, the mRNA and protein expressions of AFP and HNF4α, and the mRNA expression of ALB were significantly down-regulated in the activator Wnt3a group 0 < 0.05). Compared with hepatocyte-like differentiation group and activator Wnt3a group, the inhibitor Dkk-1 group had higher expression of AFP, HNF4α mRNA and their proteins as well as the mRNA expression of ALB (0 < 0.05). Findings from the Periodic Acid-Schiff staining, low-density lipoprotein uptake test and indocyanine green absorption test showed more positive cells in the inhibitor Dkk-1 group than in the hepatocyte-like differentiation group and least positive cells in the activator Wnt3a group. Overall, these findings suggest that the inhibition cf Wnt/β-catenin signaling pathway promotes the differentiation cf umbilical cord mesenchymal stem cells into hepatocyte-like cells; conversely, the cell differentiation can be inhibited cia the Wnt/β-catenin pathway. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Combined effects of TGFβ1 and BMP2 in serum-free chondrogenic differentiation of mesenchymal stem cells induced hyaline-like cartilage formation

This study investigated the effects of TGFβ1, BMP2 or a combination of both on the chondrogenic differentiation of mesenchymal stem cells (MSCs) in a serum-free micromass culture system in vitro. Putative MSCs harvested from the iliac crest of 4–5 month old New Zealand White Rabbits were expanded and cultured in three-dimensional high density micromass aggregate cultures containing TGFβ1, BMP2 or a combination of both, in the absence of serum. After 14–20 days of culture, chondrogenic differentiation of the MSCs was assayed by toluidine blue staining, immunohistochemistry and semi-quantitative RT-RCR of type I collagen (CI) and type II collagen (CII). Quantitative measurements of cell proliferation and sulfated glycosaminoglycan (s-GAG) were also carried out to assess the growth rate and matrix deposition of the cultured aggregates. Both immunohistochemical staining and semi-quantitative RT-PCR showed that the combination of BMP2 and TGFβ1 resulted in a marked enhancement of collagen II synthesis, with minimal collagen I expression, which would suggest hyaline-like cartilage formation. Additionally, BMP2 and TGFβ1 had a synergistic effect on matrix proteoglycan deposition, as assessed by metachromatic toluidine blue staining. This is consistent with the quantitative measurement of glycosaminoglycans, whereby a significant increase in GAG/DNA was noted in the co-treatment group. Hence, it can be concluded that the combination of BMP2 and TGFβ1 has a synergistic effect on the differentiation of MSC into hyaline-like cartilage tissue.     

Is continuous treatment with transforming growth factor-beta necessary to induce chondrogenic differentiation in mesenchymal stem cells?

This study was conducted to determine whether short-term administration of transforming growth factor (TGF)-beta would be as effective for inducing chondrogenesis in human mesenchymal stem cells (hMSCs) as continuous treatment. Four groups of hMSCs were cultured in a monolayer for 3 days followed by a pellet culture for 3 weeks under various conditions: group A, the control group, no growth factors treated; group B, 5 ng/ml of TGF-beta(2) was treated for 3 days in monolayer culture; group C, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 3 days of pellet culture; group D, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 10 days of pellet culture; group E, 5 ng/ml of TGF-beta(2) was continuously treated throughout the culture period. Glycosaminoglycan contents significantly increased in group E only. Real-time PCR indicated that expression of Sox-9, type II collagen, type II procollagen B and type X collagen increased with longer duration of TGF-beta(2) treatment. The histological findings showed that longer duration of TGF-beta(2) treatment led to significantly better quality of chondrogenesis. This study demonstrated that longer duration of TGF-beta treatment is necessary for effective chondrogenesis in hMSCs from bone marrow.     

Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration

Objective: To develop tissue engineering scaffolds consisting of self-assembling KLD-12 polypeptide/TGF-β1 nanofiber gel, for the induction of mesenchymal stem cell (MSCs) differentiation into nucleus pulposus (NP)-like cells. Methods: The release of TGF-β1 from KLD-12 polypeptide gels containing varying TGF-β1 concentrations was detected by ELISA. MSCs were isolated with a density gradient method and their differentiation into NP-like cells was analyzed in KLD-12 polypeptide/TGF-β1- or KLD-12 polypeptide control nanofiber-gel 3D-cultures. The Alcianblue method, Real-time quantitative PCR (RT-qPCR), and immunocytochemistry were used to measure the expression of extracellular matrix (ECM) molecules, such as aggrecan, glycosaminoglycans (GAGs), and type II collagen. Results: ELISA results documented favorable time-dependent release characteristics of TGF-β1 in the KLD-12 polypeptide/TGF-β1 gel scaffolds. The results of CCK-8 cell proliferation assay showed the TGF-β1 containing scaffolds induced higher growth rate in MSCs compared to the control group. Calcein-AM/PI fluorescent staining showed: the cells in the gel grew well, maintaining the circular shape of cells, and the spindle and fusiform shape of cells on the gel edges. The cell viability displayed a survival rate of 89.14% ± 2.468 for the TGF-β1 group with no significant difference between the two groups at 14 d of culture. The production of ECM was monitored showing higher expression of GAGs in the TGF-β1 group (P < 0.01) with highest amounts at 10 d and 14 d compared to 4 d and 7 d (P < 0.05). Real-time PCR results revealed that the expression levels of collagen II and aggrecan mRNA were higher in the TGF-β1 group (P < 0.05). Finally, immunocytochemical staining of collagen II confirmed the higher expression levels. Conclusion: A scaffold containing a KLD-12 polypeptide/TGF-β1-nanofiber gel and MSCs differentiated into NP-like cells is able to produce ECM and has the potential to serve as a three-dimensional (3-D) support scaffold for the filling of early postoperative residual cavities and the treatment of intervertebral disc degeneration.     

Effects of mesenchymal stem cells harboring the Interferon-β gene on A549 lung cancer in nude mice

Interferon-β (IFN-β) exhibits a tumor-killing effect; however, injection of IFN-β alone for lung cancer is often accompanied by side effects. This study investigated the possibility of using umbilical cord mesenchymal stem cells (MSCs) as cellular carriers of IFN-β.Isolated umbilical cord MSCs were transfected with a lentivirus packaging IFN-β-overexpression plasmid. A549 cells were subcutaneously injected into nude mice to establish a non-small cell lung cancer (NSCLC) mouse model. A total of 50 mice were randomly assigned to 5 different groups: a control group, IFN-β group, IFN-β-MSCs group, MSCs-lentivirus group, and MSCs group. Next, the IFN-β-MSCs, MSCs-lentivirus, and MSCs were injected into the A549 lung cancer-bearing mice in the IFN-β-MSCs, MSCs-lentivirus and MSCs groups, respectively. Mice in the control and IFN-β groups were injected with solvent or IFN-β solution. The tumors in nude mice in the IFN-β and IFN-β-MSCs groups grew at significantly slower rates than tumors in the control group, and tumors in the MSCs-lentivirus and MSC groups also grew slowly. The rates of tumor cell apoptosis in the IFN-β and IFN-β-MSCs groups were significantly higher than those in the MSCs-lentivirus and MSCs groups. The livers, lungs, and kidneys of nude mice in the IFN-β group displayed hyperemia, exudation, and pathological lesions, while those of nude mice in the IFN-β-MSCs group showed no abnormal changes. Both INF-β-MSCs and INF-β inhibited the growth of subcutaneously implanted lung tumors; however, INF-β-MSCs specifically targeted the tumor cells, and did not produce the damage to internal organs caused by the use of INF-β alone.     

Matrix stiffness determines the fate of nucleus pulposus–derived stem cells

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus–derived stem cells (NP–SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G′ = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G′ = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.     

Determination of dual delivery for stem cell differentiation using dexamethasone and TGF-β3 in/on polymeric microspheres

In this work, a cocktail of dexamethasone (Dex) and transforming growth factor-β3 (TGF-β3) bound with heparin in/on PLGA microspheres was investigated by local dual delivery using rabbit mesenchymal stem cells (rMSCs) for chondrogenic differentiation in in vitro and in vivo study. In this study, we proved that the microsphere constructs were capable of simultaneously releasing TGF-β3-conjugated with cy5.5 and Dex-conjugated with FITC with approximately zero order kinetics determined by Kodak imaging and confocal laser microscope. Microsphere constructs containing TGF-β3 and Dex showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study irrespective of the presence of dexamethasone and TGF-β3. Thus, dual delivery of TGF and Dex can be used to engineer inflammation-free and cartilage-associate tissue in the transplanted PLGA construct into nude mouse. These heparin-bound TGF-β3-coated and Dex-loaded PLGA microsphere constructs show promise as coatings for implantable biomedical devices to improve biocompatibility and ensure both in vitro and in vivo performance.     

Effect of substrate stiffness on pulmonary fibroblast activation by TGF-β

Peptide crosslinkers containing the sequence C-X-CG (X represents various adhesive peptides) were incorporated into poly(ethylene glycol) (PEG) hydrogel networks with different mechanical properties. Pulmonary fibroblasts (PFs) exhibit increased adhesion to rigid hydrogels modified with X=RGDS, DGEA and IKVAV (0.5 and/or 5 mM) compared with a scrambled control (X=HRPNS). PFs exhibit increased adhesion to softer hydrogels when X=DGEA at low (0.5 mM) peptide concentration. PFs seeded onto hydrogels modified with X=RGDS produce alpha-smooth muscle actin (α-SMA), a myofibroblast marker, and form an extensive cytoskeleton with focal adhesions. Decreasing substrate stiffness (achieved through hydrolytic degradation) results in down-regulation of α-SMA expression by PFs. Substrate stiffness increases the sensitivity of PFs to exogenously applied transforming growth factor beta (TGF-β1); PFs on the most rigid gels (E=900 kPa) express α-SMA when treated with low concentrations of TGF-β1 (1 ng ml(-1)), while those on less rigid gels (E=20-60 kPa) do not. These results demonstrate the importance of both mechanical and chemical cues in studying pulmonary fibroblast activation, and establish PEG hydrogels as a viable material for further study of IPF etiology.     

The role of physiological mechanical cues on mesenchymal stem cell differentiation in an airway tract-like dense collagen–silk fibroin construct

Airway tracts serve as a conduit of transport in the respiratory system. Architecturally, these are composed of cartilage rings that offer flexibility and prevent collapse during normal breathing. To this end, the successful regeneration of an airway tract requires the presence of differentiated chondrocytes and airway smooth muscle cells. This study investigated the role of physiological dynamic mechanical stimulation, in vitro, on the differentiation of mesenchymal stem cells (MSCs), three-dimensionally seeded within a tubular dense collagen matrix construct-reinforced with rings of electrospun silk fibroin mat (TDC–SFC). In particular, the role of either shear stress supplied by laminar fluid flow or cyclic shear stress in combination with circumferential strain, provided by pulsatile flow, on the chondrogenic differentiation, and contractile lineage of MSCs, and their effects on TDC–SFC morphology and mechanical properties were analysed. Chondrogenic differentiation of MSCs was observed in the presence of chondrogenic supplements under both static and laminar flow cultures. In contrast, physiological pulsatile flow resulted in preferential cellular orientation within TDC–SFC, as dictated by dynamic circumferential strain, and induced MSC contractile phenotype expression. In addition, pulsatile flow decreased MSC-mediated collagen matrix remodelling and increased construct circumferential strength. Therefore, TDC–SFC demonstrated the central role of a matrix in the delivery of mechanical stimuli over chemical factors, by providing an in vitro niche to control MSC differentiation, alignment and its capacity to remodel the matrix.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

The effect of blood phenylalanine concentration on Kuvan™ response in phenylketonuria

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH) with consequent elevation of blood phenylalanine (Phe), reduction in tyrosine (Tyr) and elevation of Phe/Tyr ratio (P/T). Although newborn screening for PKU with early dietary treatment improved severe, irreversible brain damage, older patients suffer reversible losses in executive function when Phe concentrations are elevated. The maintenance of strict nutritional control in older children and adults remains difficult. An adjunct to dietary therapy, oral tetrahydrobiopterin (BH₄) has recently been approved by the Food and Drug Administration as a stable, synthetic BH₄ called Kuvan?. Published studies of Kuvan response in PKU varies and involved primarily children. In this prospective study we evaluated dose–response, response frequency and factors predicting response in 21 patients with PKU (aged 8–30 years), who required life-long dietary treatment. Response to Kuvan was defined at 24 h (acute) and over 4 weeks (chronic) as a ≥ 30% decline in the Phe or P/T ratio. A dose of 20 mg/kg Kuvan was chosen with 29% responding in 24 h and 33% of patients at 4 weeks. We then compared baseline Phe, Tyr, P/T, Phe intake, and frequency of “severe” versus “moderate” mutant PAH alleles among acute and chronic responders and non-responders to Kuvan. Predictors of response to Kuvan, both acute and chronic were baseline Phe and baseline P/T. Baseline Phe and P/T were higher among non-responders (P < 0.05). By contrast baseline Tyr was similar (P = 0.45). Phe intake tended to be higher (18 ± 20 mg/kg/24 h) among Kuvan responders than non-responders (15 ± 11 mg/kg/24 h), P < 0.07 NS. Similarly the frequency of “severe” mutant PAH alleles tended to be more frequent (67%) among non-responders than responders (40%) by Chi² test, P = 0.08 NS. These results were reproducible in a “responder” to Kuvan. To assess directly the effect of elevated blood Phe, Phe was lowered in four, “non-responder” patients, but all failed to respond to Kuvan. We conclude that baseline blood Phe and P/T ratio can predict increased probability for response to Kuvan by patients with classic PKU, but the in vivo mechanisms of response to Kuvan remain enigmatic.