Biomineralization, mechanical, antibacterial and biological investigation of larnite and rankinite bioceramics.

This work is aimed to develop a biocompatible, bactericidal and mechanically stable biomaterial to overcome the challenges associated with calcium phosphate bioceramics. The influence of chemical composition on synthesis temperature, bioactivity, antibacterial activity and mechanical stability of least explored calcium silicate bioceramics was studied. The current study also investigates the biomedical applications of rankinite (Ca3Si2O7) for the first time. Sol-gel combustion method was employed for their preparation using citric acid as a fuel. Differential thermal analysis indicated that the crystallization of larnite and rankinite occurred at 795 °C and 1000 °C respectively. The transformation of secondary phases into the desired product was confirmed by XRD and FT-IR. TEM micrographs showed the particle size of larnite in the range of 100-200 nm. The surface of the samples was entirely covered by the dominant apatite phase within one week of immersion. Moreover, the compressive strength of larnite and rankinite was found to be 143 MPa and 233 MPa even after 28 days of soaking in SBF. Both samples prevented the growth of clinical pathogens at a concentration of 2 mg/mL. Larnite and rankinite supported the adhesion, proliferation and osteogenic differentiation of hBMSCs. The variation in chemical composition was found to influence the properties of larnite and rankinite. The results observed in this work signify that these materials not only exhibit faster biomineralization ability, excellent cytocompatibility but also enhanced mechanical stability and antibacterial properties.

Mechanical Strain-Mediated Tenogenic Differentiation of Mesenchymal Stromal Cells Is Regulated through Epithelial Sodium Channels.

It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, ß, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.

In vitro evaluation of novel low-pressure spark plasma sintered HA-BG composite scaffolds for bone tissue engineering.

The low-pressure spark plasma sintering (SPS) technique is adopted to fabricate hydroxyapatite-bioglass (HA-BG) scaffolds while maintaining the physical properties of both components, including their bulk and relative density and hardness. However, prior to their orthopaedic and dental applications, these scaffolds must be validated via pre-clinical assessments. In the present study, scaffolds with different ratios of HA : BG, namely, 100 : 0 (HB 0 S), 90 : 10 (HB 10 S), 80 : 20 (HB 20 S) and 70 : 30 (HB 30 S) were fabricated. These scaffolds were characterized by investigating their physicochemical properties (X-ray diffraction (XRD) and surface wettability), bioactivity in a simulated body fluid (SBF) (field emission scanning electron microscopy (FESEM), Fourier-transform infrared spectroscopy (FTIR) and calcium dissolution), antimicrobial properties, biocompatibility and osteoinduction of human bone marrow-derived mesenchymal stromal cells (hBMSCs) and human monocyte immune cell response. The XRD and surface wettability results confirmed no formation of undesirable phases and the enhanced surface hydrophilicity of the scaffolds, respectively. The bioactivity in SBF indicated the formation of bone-like apatite on the surface of the scaffolds, corresponding to an increase in BG%, which was confirmed through FTIR spectra and the increasing trend of calcium release in SBF. The scaffolds showed inhibition properties against Staphylococcus aureus and Staphylococcus epidermidis. The scanning electron microscopy (SEM) micrographs and Alamar Blue proliferation assay indicated the good attachment and significant proliferation, respectively, of hBMSCs on the scaffolds. Alizarin Red S staining confirmed that the scaffolds supported the mineralisation of hBMSCs. The osteogenic protein secretion (bone morphogenetic protein-2 (BMP2), type-I collagen (COL1) and osterix (OSX)) was significant on the HB 30 S-seeded hBMSCs when compared with that of HB 0 S. The monocyte migration was significantly halted in response to HA-BG-conditioned media when compared with the positive control (monocyte chemoattractant protein-1: MCP-1). In conclusion, the HB 30 S composite scaffold has a greater potential to substitute bone grafts in orthopaedic and dental applications.

Effect of Chitosan Nanoparticle-Loaded Thymus serpyllum on Hydrogen Peroxide-Induced Bone Marrow Stromal Cell Damage.

We have determined the protective effects of Thymus serpyllum (TS) extract and nanoparticle-loaded TS on hydrogen peroxide-induced cell death of mesenchymal stromal cells (MSCs) in vitro. Gas chromatography-mass spectroscopy confirmed the spectrum of active components in the extract. Out of the three different extracts, the hexane extract showed significant free radical scavenging activity. Treatment of MSCs with H2O2 (hydrogen peroxide) significantly increased intracellular cell death; however, pretreatment with TS extract and nanoparticle-loaded TS (200 µg/ml) suppressed H2O2-induced elevation of Cyt-c and MMP13 and increased the survival rates of MSCs. H2O2-induced (0.1 mM) changes in cytokines were attenuated in the extract and nanoparticles by pretreatment and cotreatment at two time points (p < 0.05). H2O2 increased cell apoptosis. In contrast, treatment with nanoparticle-loaded TS suppressed the percentage of apoptosis considerably (p < 0.05). Therefore, TS may be considered as a potential candidate for enhancing the effectiveness of MSC transplantation in cell therapy.

The physicochemical and biomechanical profile of forsterite and its osteogenic potential of mesenchymal stromal cells.

It has been demonstrated that nanocrystalline forsterite powder synthesised using urea as a fuel in sol-gel combustion method had produced a pure forsterite (FU) and possessed superior bioactive characteristics such as bone apatite formation and antibacterial properties. In the present study, 3D-scaffold was fabricated using nanocrystalline forsterite powder in polymer sponge method. The FU scaffold was used in investigating the physicochemical, biomechanics, cell attachment, in vitro biocompatibility and osteogenic differentiation properties. For physicochemical characterisation, Fourier-transform infrared spectroscopy (FTIR), Energy dispersive X-ray (EDX), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoemission spectrometer (XPS) and Brunauer-Emmett-Teller (BET) were used. FTIR, EDX, XRD peaks and Raman spectroscopy demonstrated correlating to FU. The XPS confirmed the surface chemistry associating to FU. The BET revealed FU scaffold surface area of 12.67 m2/g and total pore size of 0.03 cm3/g. Compressive strength of the FU scaffold was found to be 27.18 ± 13.4 MPa. The human bone marrow derived mesenchymal stromal cells (hBMSCs) characterisation prior to perform seeding on FU scaffold verified the stromal cell phenotypic and lineage commitments. SEM, confocal images and presto blue viability assay suggested good cell attachment and proliferation of hBMSCs on FU scaffold and comparable to a commercial bone substitutes (cBS). Osteogenic proteins and gene expression from day 7 onward indicated FU scaffold had a significant osteogenic potential (p<0.05), when compared with day 1 as well as between FU and cBS. These findings suggest that FU scaffold has a greater potential for use in orthopaedic and/or orthodontic applications.

Genetically modified mesenchymal stem/stromal cells transfected with adiponectin gene can stably secrete adiponectin.

AIMS: Mesenchymal stem/stromal cells (MSCs) hold promises for the treatment of diverse diseases and regeneration of injured tissues. Genetic modification of MSCs through gene delivery might enhance their therapeutic potential. Adiponectin has been appeared as a potential biomarker for predicting various diseases. Plasma adiponectin levels are negatively correlated with various metabolic and vascular diseases and supplementation of exogenous adiponectin ameliorates the diseases. This study aims to develop adiponectin secreting genetically modified MSCs (GM-MSCs) as a potent strategic tool to complement endogenous adiponectin for the treatment of adiponectin deficiency diseases. MAIN METHODS: Human bone marrow derived MSCs were isolated, expanded in vitro and transfected with adiponectin gene containing plasmid vector. Total RNA was extracted and cDNA was prepared by reverse transcription polymerase chain reaction (RT-PCR). The expression of adiponectin gene and protein in GM-MSCs was analyzed by PCR and Western blotting respectively. The secretion of adiponectin protein from GM-MSCs was analyzed by enzyme-linked immunosorbent assay. KEY FINDINGS: The expression of adiponectin gene and plasmid DNA was detected in GM-MSCs but not in control group of MSCs. Adiponectin gene expression was detected in GM-MSCs at 2, 7, 14, 21 and 28days after transfection. Western blotting analysis revealed the expression of adiponectin protein only in GM-MSCs. The GM-MSCs stably secreted adiponectin protein into culture media at least for 4weeks. SIGNIFICANCE: GM-MSCs express and secret adiponectin protein. Therefore, these adiponectin secreting GM-MSCs could be instrumental for the supplementation of adiponectin in the treatment of adiponectin deficiency related diseases.

Differential cytotoxic activity of Quercetin on colonic cancer cells depends on ROS generation through COX-2 expression.

Quercetin is a bioactive compound with anti-inflammatory, antioxidant and anticancer properties. This study exemplifies the differential cytotoxic activity of Quercetin on two human colonic cancer cell lines, HT29 and HCT15. IC50 of Quercetin for HT29 and HCT15 cells were 42.5 µM and 77.4 µM, respectively. Activation of caspase-3, increased level of cytosolic cytochrome c, decreased levels of pAkt, pGSK-3ß and cyclin D1 in 40 µM Quercetin treated HT29 cells alone. Though, nuclear translocation of NFkB was increased in 40 µM Quercetin treated HT29 and HCT15 cells, over expression of COX-2 was observed in 40 µM Quercetin treated HT29 cells, whereas, Quercetin treated HCT15 cells did not expressed COX-2. Increased generation of reactive oxygen species (ROS) was observed only in Quercetin treated HT29 cells, which is due to over expression of COX-2, as COX-2 silencing inhibited Quercetin induced apoptosis and ROS generation. Insilico analysis provided evidence that Quercetin could partially inhibit COX-2 enzyme by binding to subunit A which has peroxidase activity and serves as source of ROS. However, Quercetin showed minimal effect on normal intestinal epithelial cells i,e IEC-6. To conclude, differential sensitivity of two cancer cells, HT29 and HCT15, to Quercetin depends on COX-2 dependent ROS generation that induces apoptosis and inhibits cell survival.

Incorporation of Human-Platelet-Derived Growth Factor-BB Encapsulated Poly(lactic-co-glycolic acid) Microspheres into 3D CORAGRAF Enhances Osteogenic Differentiation of Mesenchymal Stromal Cells.

Tissue engineering aims to generate or facilitate regrowth or healing of damaged tissues by applying a combination of biomaterials, cells, and bioactive signaling molecules. In this regard, growth factors clearly play important roles in regulating cellular fate. However, uncontrolled release of growth factors has been demonstrated to produce severe side effects on the surrounding tissues. In this study, poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) incorporated three-dimensional (3D) CORAGRAF scaffolds were engineered to achieve controlled release of platelet-derived growth factor-BB (PDGF-BB) for the differentiation of stem cells within the 3D polymer network. Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, scanning electron microscopy, and microtomography were applied to characterize the fabricated scaffolds. In vitro study revealed that the CORAGRAF-PLGA-PDGF-BB scaffold system enhanced the release of PDGF-BB for the regulation of cell behavior. Stromal cell attachment, viability, release of osteogenic differentiation markers such as osteocalcin, and upregulation of osteogenic gene expression exhibited positive response. Overall, the developed scaffold system was noted to support rapid cell expansion and differentiation of stromal cells into osteogenic cells in vitro for bone tissue engineering applications.

Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

In-vitro bioactivity, biocompatibility and dissolution studies of diopside prepared from biowaste by using sol-gel combustion method.

Diopside was synthesized from biowaste (Eggshell) by sol-gel combustion method at low calcination temperature and the influence of two different fuels (urea, l-alanine) on the phase formation temperature, physical and biological properties of the resultant diopside was studied. The synthesized materials were characterized by heating microscopy, FTIR, XRD, BET, SEM and EDAX techniques. BET analysis reveals particles were of submicron size with porosity in the nanometer range. Bone-like apatite deposition ability of diopside scaffolds was examined under static and circulation mode of SBF (Simulated Body Fluid). It was noticed that diopside has the capability to deposit HAP (hydroxyapatite) within the early stages of immersion. ICP-OES analysis indicates release of Ca, Mg, Si ions and removal of P ions from the SBF, but in different quantities from diopside scaffolds. Cytocompatability studies on human bone marrow stromal cells (hBMSCs) revealed good cellular attachment on the surface of diopside scaffolds and formation of extracellular matrix (ECM). This study suggests that the usage of eggshell biowaste as calcium source provides an effective substitute for synthetic starting materials to fabricate bioproducts for biomedical applications.

The effect of strontium ranelate on the healing of a fractured ulna with bone gap in rabbit.

BACKGROUND: Fracture healing in bone gap is one of the major challenges encountered in Orthopedic Surgery. At present, the treatment includes bone graft, employing either internal or external fixation which has a significant impact on the patient, family and even society. New drugs are emerging in the markets such as anabolic bone-forming agents including teriparatide and strontium ranelate to stimulate bone growth. Based on the mechanism of their actions, we embarked on a study on the healing of a fractured ulna with bone gap in a rabbit model. We segregated ten rabbits into two groups: five rabbits in the test group and five rabbits in the control group. We created a 5 mm bone gap in the ulna bone, removing the periosteum as well. Rabbits in the test group received 450 mg/kg of strontium ranelate via oral administration, daily, for six weeks. The x-rays, CT scans and blood tests were performed every two weeks. At the end of six weeks, the rabbits were sacrificed, and the radius and ulna bones harvested for histopathological examination. RESULTS: Based on the x-rays and CT scans, fracture healing or bone formation was observed to be faster in the control group. From the x-ray findings, 80 % of the fracture united and by CT scan, 60 % of the fracture united in the control group at the end of the six-week study. None of the fractures united in the test group. However, the histopathology report showed that a callus of different stages was being formed in both groups, consisting of 80 % of bone. The serum levels of osteocalcin and alkaline phosphatase initially remained similar up to three weeks and changed slightly at the end of six weeks. CONCLUSIONS: We conclude that the strontium effect begins slowly, and while it may not interfere with bone cell proliferation it may interfere in the mineralization and delay the acute stage of fracture healing. We recommend that a larger sample size and a longer duration of the study period be implemented to confirm our finding.

Three dimensional alginate-fucoidan composite hydrogel augments the chondrogenic differentiation of mesenchymal stromal cells.

Presence of sulfated polysaccharides like heparan sulphate has often been implicated in the regulation of chondrogenesis. However, recently there has been a plethora of interest in the use of non-animal extracted analogs of heparan sulphate. Here we remodeled alginate (1.5%) by incorporating fucoidan (0.5%), a natural sulphated polysaccharide extracted from seaweeds to form a composite hydrogel (Al-Fu), capable of enhancing chondrogenesis of human mesenchymal stromal cells (hMSCs). We confirmed the efficiency of fucoidan incorporation by FTIR and EDX analysis. Further, its ability to support hMSC attachment and chondrogenic differentiation was confirmed by SEM, biochemical glycosaminoglycan quantification, real-time quantitative PCR and immunocytochemical analyses of chondrogenic markers Sox-9, Collagen II, Aggrecan and COMP. Effect of Al-Fu hydrogel on hMSC hypertrophy was also confirmed by the downregulation of hypertrophic genes Collagen X and Runx2. This composite scaffold can hence be used as a cartilage biomimetic biomaterial to drive hMSC chondrogenesis and for other cartilage repair based therapies.

Incorporation of Fucoidan in ß-Tricalcium phosphate-Chitosan scaffold prompts the differentiation of human bone marrow stromal cells into osteogenic lineage.

In our previous study, we reported the fabrication and characterization of a novel tricalcium phosphate-fucoidan-chitosan (TCP-Fu-Ch) biocomposite scaffold. However, the previous report did not show whether the biocomposite scaffold can exhibit osteogenic differentiation of human bone marrow stromal cells in osteogenic media and normal media supplemented with platelet-derived growth factor (PDGF-BB). On day 15, the release of osteocalcin, was significant in the TCP-Fu-Ch scaffold, when compared with that in the TCP-Ch scaffold, and the level of release was approximately 8 and 6 ng/ml in osteogenic and normal media supplemented with PDGF-BB, respectively. Scanning electron microscopy of the TCP-Fu-Ch scaffold demonstrated mineralization and apatite layer formation on day 14, while the addition of PDGF-BB also improved the osteogenic differentiation of the scaffold. An array of gene expression analysis demonstrated that TCP-Fu-Ch scaffold cultured in osteogenic and normal media supplemented with PDGF-BB showed significant improvement in the expression of collagen 1, Runt-related transcription factor 2, osteonectin, bone gamma-carboxyglutamate protein, alkaline phosphatase, and PPA2, but a decline in the expression of integrin. Altogether, the present study demonstrated that fucoidan-incorporated TCP-Ch scaffold could be used in the differentiation of bone marrow stromal cells and can be a potential candidate for the treatment of bone-related ailments through tissue engineering technology.

Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads.

Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering.

Fabrication and in vitro biological activity of ßTCP-Chitosan-Fucoidan composite for bone tissue engineering.

We developed tricalcium phosphate-chitosan-fucoidan biocomposite scaffold (TCP-Ch-Fu) by using the freeze-drying technique. The fabricated biocomposite scaffolds were analyzed by spectroscopy and porosity measurement. The biomechanical properties of scaffolds were assessed by compression test and the results suggested that the incorporation of Fucoidan into the biocomposite improves the compression strength of scaffolds. Biomineralization of scaffolds was evaluated by soaking them in simulated body fluid and the results revealed that the addition of Fucoidan into the scaffolds enhanced the formation of apatite layer on the surface of biocomposite after 7 days of immersion. Alamar Blue assay confirmed that the cell viability of human-derived bone marrow stromal cell was superior in the TCP-Ch-Fuscaffold. The addition of Fucoidan to TCP-Ch increased the release of osteocalcin, confirming that it can support osteogenic differentiation of human mesenchymal stromal cells in in vitro culture. Thus, TCP-Ch-Fu could be a potential candidate for bone-tissue engineering applications.

Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold.

AIM: To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold. MATERIALS AND METHODS: The porosity and pore size were analyzed using micro-CT. The biocompatibility was demonstrated by alamar blue assay, and cell attachment through SEM and Hoechst staining. The osteogenic differentiation was demonstrated using biochemical assay and osteogenic gene expression. RESULTS: BDHA and CHA scaffolds showed porosity of 76.6 ± 0.6 and 64.3 ± 0.3% and pore size diameter of 0.04-0.25 and 0.1-2.6 mm, respectively. hMSCs proliferation, ALP activity, osteocalcin secretion and osteogenic gene expression are comparable in both the scaffolds. CONCLUSION: These results demonstrated that BDHA is biocompatible, supports cell adhesion and promotes proliferation and osteogenic differentiation.

PVA-chitosan composite hydrogel versus alginate beads as a potential mesenchymal stem cell carrier for the treatment of focal cartilage defects.

PURPOSE: To investigate whether mesenchymal stem cells (MSCs) seeded in novel polyvinyl alcohol (PVA)-chitosan composite hydrogel can provide comparable or even further improve cartilage repair outcomes as compared to previously established alginate-transplanted models. METHODS: Medial femoral condyle defect was created in both knees of twenty-four mature New Zealand white rabbits, and the animals were divided into four groups containing six animals each. After 3 weeks, the right knees were transplanted with PVA-chitosan-MSC, PVA-chitosan scaffold alone, alginate-MSC construct or alginate alone. The left knee was kept as untreated control. Animals were killed at the end of 6 months after transplantation, and the cartilage repair was assessed through Brittberg morphological score, histological grading by O'Driscoll score and quantitative glycosaminoglycan analysis. RESULTS: Morphological and histological analyses showed significant (p < 0.05) tissue repair when treated with PVA-chitosan-MSC or alginate MSC as compared to the scaffold only and untreated control. In addition, safranin O staining and the glycosaminoglycan (GAG) content were significantly higher (p < 0.05) in MSC treatment groups than in scaffold-only or untreated control group. No significant difference was observed between the PVA-chitosan-MSC- and alginate-MSC-treated groups. CONCLUSION: PVA-chitosan hydrogel seeded with mesenchymal stem cells provides comparable treatment outcomes to that of previously established alginate-MSC construct implantation. This study supports the potential use of PVA-chitosan hydrogel seeded with MSCs for clinical use in cartilage repair such as traumatic injuries.

Platelet rich concentrate promotes early cellular proliferation and multiple lineage differentiation of human mesenchymal stromal cells in vitro.

Platelet rich concentrate (PRC) is a natural adjuvant that aids in human mesenchymal stromal cell (hMSC) proliferation in vitro; however, its role requires further exploration. This study was conducted to determine the optimal concentration of PRC required for achieving the maximal proliferation, and the need for activating the platelets to achieve this effect, and if PRC could independently induce early differentiation of hMSC. The gene expression of markers for osteocytes (ALP, RUNX2), chondrocytes (SOX9, COL2A1), and adipocytes (PPAR-γ) was determined at each time point in hMSC treated with 15% activated and nonactivated PRC since maximal proliferative effect was achieved at this concentration. The isolated PRC had approximately fourfold higher platelet count than whole blood. There was no significant difference in hMSC proliferation between the activated and nonactivated PRC. Only RUNX2 and SOX9 genes were upregulated throughout the 8 days. However, protein expression study showed formation of oil globules from day 4, significant increase in ALP at days 6 and 8 (P ≤ 0.05), and increased glycosaminoglycan levels at all time points (P < 0.05), suggesting the early differentiation of hMSC into osteogenic and adipogenic lineages. This study demonstrates that the use of PRC increased hMSC proliferation and induced early differentiation of hMSC into multiple mesenchymal lineages, without preactivation or addition of differentiation medium.

Biomechanical evaluation of two commonly used external fixators in the treatment of open subtalar dislocation--a finite element analysis.

Subtalar dislocation is a rare injury caused by high-energy trauma. Current treatment strategies include leg casts, internal fixation and external fixation. Among these, external fixators are the most commonly used as this method is believed to provide better stabilization. However, the biomechanical stability provided by these fixators has not been demonstrated. This biomechanical study compares two commonly used external fixators, i.e. Mitkovic and Delta. CT imaging data were used to reconstruct three-dimensional models of the tibia, fibula, talus, calcaneus, navicular, cuboid, three cuneiforms and five metatarsal bones. The 3D models of the bones and cartilages were then converted into four-noded linear tetrahedral elements, whilst the ligaments were modelled with linear spring elements. Bones and cartilage were idealized as homogeneous, isotropic and linear. To simulate loading during walking, axial loading (70 N during the swing and 350 N during the stance phase) was applied at the end of diaphyseal tibia. The results demonstrate that the Mitkovic fixator produced greater displacement (peak 3.0mm and 15.6mm) compared to the Delta fixator (peak 0.8mm and 3.9 mm), in both the swing and stance phase, respectively. This study demonstrates that the Delta external fixator provides superior stability over the Mitkovic fixator. The Delta fixator may be more effective in treating subtalar dislocation.