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.

Platelet-rich concentrate in serum-free medium enhances cartilage-specific extracellular matrix synthesis and reduces chondrocyte hypertrophy of human mesenchymal stromal cells encapsulated in alginate.

Platelet-rich concentrate (PRC), used in conjunction with other chondroinductive growth factors, have been shown to induce chondrogenesis of human mesenchymal stromal cells (hMSC) in pellet culture. However, pellet culture systems promote cell hypertrophy and the presence of other chondroinductive growth factors in the culture media used in previous studies obscures accurate determination of the effect of platelet itself in inducing chondrogenic differentiation. Hence, this study aimed to investigate the effect of PRC alone in enhancing the chondrogenic differentiation potential of human mesenchymal stromal cells (hMSC) encapsulated in three-dimensional alginate constructs. Cells encapsulated in alginate were cultured in serum-free medium supplemented with only 15% PRC. Scanning electron microscopy was used to determine the cell morphology. Chondrogenic molecular signature of hMSCs was determined by quantitative real-time PCR and verified at protein levels via immunohistochemistry and enzyme-linked immunosorbent assay. Results showed that the cells cultured in the presence of PRC for 24 days maintained a chondrocytic phenotype and demonstrated minimal upregulation of cartilaginous extracellular matrix (ECM) marker genes (SOX9, TNC, COL2, ACAN, COMP) and reduced expression of chondrocyte hypertrophy genes (Col X, Runx2) compared to the standard chondrogenic medium (p < 0.05). PRC group had correspondingly higher levels of glycosaminoglycan and increased concentration of chondrogenic specific proteins (COL2, ACAN, COMP) in the ECM. In conclusion, PRC alone appears to be very potent in inducing chondrogenic differentiation of hMSCs and offers additional benefit of suppressing chondrocyte hypertrophy, rendering it a promising approach for providing abundant pool of chondrogenic MSCs for application in cartilage tissue engineering.

Cancer stem cells and evolving novel therapies: a paradigm shift.

Accumulating evidence of stem-like cells/cancer stem cells (CSCs) has been gaining attention of cancer researchers over the last decade. Though many tumors harbor CSCs in their dedicated niches, identifying and exterminating those cells has proved to be difficult, due to their heterogenous nature, as the CSC phenotype vary substantially and may undergo reversible phenotypic changes. As a tumor propagation initiator, CSCs are considered as an exciting novel therapy for a better therapeutic outcome. This review discusses the major advances in the development of CSC-based therapies of most common cancers which includes lung, cervix and liver cancers.

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.

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.

Cytotoxic evaluation of hydroxyapatite-filled and silica/hydroxyapatite-filled acrylate-based restorative composite resins: An in vitro study.

STATEMENT OF PROBLEM: Although the physical and mechanical properties of hydroxyapatite-filled dental restorative composite resins have been examined, the biocompatibility of these materials has not been studied in detail. PURPOSE: The purpose of this in vitro study was to analyze the toxicity of acrylate-based restorative composite resins filled with hydroxyapatite and a silica/hydroxyapatite combination. MATERIAL AND METHODS: Five different restorative materials based on bisphenol A-glycidyl methacrylate (bis-GMA) and tri-ethylene glycol dimethacrylate (TEGDMA) were developed: unfilled (H0), hydroxyapatite-filled (H30, H50), and silica/hydroxyapatite-filled (SH30, SH50) composite resins. These were tested for in vitro cytotoxicity by using human bone marrow mesenchymal stromal cells. Surface morphology, elemental composition, and functional groups were determined by scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), and Fourier-transformed infrared spectroscopy (FTIR). The spectra normalization, baseline corrections, and peak integration were carried out by OPUS v4.0 software. RESULTS: Both in vitro cytotoxicity results and SEM analysis indicated that the composite resins developed were nontoxic and supported cell adherence. Elemental analysis with EDX revealed the presence of carbon, oxygen, calcium, silicon, and gold, while the presence of methacrylate, hydroxyl, and methylene functional groups was confirmed through FTIR analysis. CONCLUSIONS: The characterization and compatibility studies showed that these hydroxyapatite-filled and silica/hydroxyapatite-filled bis-GMA/TEGDMA-based restorative composite resins are nontoxic to human bone marrow mesenchymal stromal cells and show a favorable biologic response, making them potential biomaterials.

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.

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.

Geometric variable designs of cam/post mechanisms influence the kinematics of knee implants.

PURPOSE: Reproducing the femoral rollback through specially designed mechanism in knee implants is required to achieve full knee function in total knee arthroplasty. Most contemporary implants use cam/post mechanism to replace the function of Posterior Cruciate Ligament. This study was aimed to determine the most appropriate cam and post designs to produce normal femoral rollback of the knee. METHODS: Three different cams (triangle, ellipse, and circle) and three different posts (straight, convex, concave) geometries were considered in this study and were analysed using kinematic analyses. Femoral rollback did not occur until reaching 50° of knee flexion. Beyond this angle, two of the nine combinations demonstrate poor knee flexion and were eliminated from the study. RESULTS: The combination of circle cam with concave post, straight post and convex post showed 15.6, 15.9 and 16.1 mm posterior translation of the femur, respectively. The use of ellipse cam with convex post and straight post demonstrated a 15.3 and 14.9 mm femoral rollback, whilst the combination of triangle cam with convex post and straight post showed 16.1 and 15.8 mm femoral rollback, respectively. CONCLUSION: The present study demonstrates that the use of circle cam and convex post created the best femoral rollback effect which in turn produces the highest amount of knee flexion. The findings of the study suggest that if the design is applied for knee implants, superior knee flexion may be possible for future patients. LEVEL OF EVIDENCE: IV.

A comparative study on in vitro osteogenic priming potential of electron spun scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for tissue engineering application.

A comparative study on the in vitro osteogenic potential of electrospun poly-L-lactide/hydroxyapatite/collagen (PLLA/HA/Col, PLLA/HA, and PLLA/Col) scaffolds was conducted. The morphology, chemical composition, and surface roughness of the fibrous scaffolds were examined. Furthermore, cell attachment, distribution, morphology, mineralization, extracellular matrix protein localization, and gene expression of human mesenchymal stromal cells (hMSCs) differentiated on the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA were also analyzed. The electrospun scaffolds with a diameter of 200-950 nm demonstrated well-formed interconnected fibrous network structure, which supported the growth of hMSCs. When compared with PLLA/H%A and PLLA/Col scaffolds, PLLA/Col/HA scaffolds presented a higher density of viable cells and significant upregulation of genes associated with osteogenic lineage, which were achieved without the use of specific medium or growth factors. These results were supported by the elevated levels of calcium, osteocalcin, and mineralization (P<0.05) observed at different time points (0, 7, 14, and 21 days). Furthermore, electron microscopic observations and fibronectin localization revealed that PLLA/Col/HA scaffolds exhibited superior osteoinductivity, when compared with PLLA/Col or PLLA/HA scaffolds. These findings indicated that the fibrous structure and synergistic action of Col and nano-HA with high-molecular-weight PLLA played a vital role in inducing osteogenic differentiation of hMSCs. The data obtained in this study demonstrated that the developed fibrous PLLA/Col/HA biocomposite scaffold may be supportive for stem cell based therapies for bone repair, when compared with the other two scaffolds.

Current knowledge on alleviating Helicobacter pylori infections through the use of some commonly known natural products: bench to bedside.

Helicobacter pylori, a spiral-shaped Gram-negative bacterium, has been classified as a class I carcinogen by the World Health Organization and recognized as the causative agent for peptic ulcers, duodenal ulcer, gastritis, mucosa-associated lymphoid tissue lymphomas, and gastric cancer. Owing to their alarming rate of drug resistance, eradication of H. pylori remains a global challenge. Triple therapy consisting of a proton pump inhibitor, clarithromycin, and either amoxicillin or metronidazole, is generally the recommended standard for the treatment of H. pylori infection. Complementary and alternative medicines have a long history in the treatment of gastrointestinal ailments and various compounds has been tested for anti-H. pylori activity both in vitro and in vivo; however, their successful use in human clinical trials is sporadic. Hence, the aim of this review is to analyze the role of some well-known natural products that have been tested in clinical trials in preventing, altering, or treating H. pylori infections. Whereas some in vitro and in vivo studies in the literature have demonstrated the successful use of a few potential natural products for the treatment of H. pylori-related infections, others indicate a need to consider natural products, with or without triple therapy, as a useful alternative in treating H. pylori-related infections. Thus, the reported mechanisms include killing of H. pylori urease inhibition, induction of bacterial cell damage, and immunomodulatory effect on the host immune system. Furthermore, both in vitro and in vivo studies have demonstrated the successful use of some potential natural products for the treatment of H. pylori-related infections. Nevertheless, the routine prescription of potential complementary and alternative medicines continues to be restrained, and evidence on the safety and efficacy of the active compounds remains a subject of ongoing debate.

The effects of staged intra-articular injection of cultured autologous mesenchymal stromal cells on the repair of damaged cartilage: a pilot study in caprine model.

INTRODUCTION: Treatment of chondral injuries remains a major issue despite the many advances made in cartilage repair techniques. Although it has been postulated that the use of marrow stimulation in combination with cell-based therapy may provide superior outcome, this has yet to be demonstrated. A pilot study was thus conducted to determine if bone marrow derived mesenchymal stromal cells (BM-MSCs) have modulatory effects on the repair outcomes of bone marrow stimulation (BMS) techniques. METHODS: Two full-thickness chondral 5 mm diameter defects were created in tandem on the medial condyle of left stifle joints of 18 Boer caprine (N = 18). Goats were then divided equally into three groups. Simultaneously, bone marrow aspirates were taken from the iliac crests from the goats in Group 1 and were sent for BM-MSC isolation and expansion in vitro. Six weeks later, BMS surgery, which involves subchondral drilling at the defect sites, was performed. After two weeks, the knees in Group 1 were given autologous intra-articular BM-MSCs (N = 6). In Group 2, although BMS was performed there were no supplementations provided. In Group 3, no intervention was administered. The caprines were sacrificed after six months. Repairs were evaluated using macroscopic assessment through the International Cartilage Repair Society (ICRS) scoring, histologic grading by O'Driscoll score, biochemical assays for glycosaminoglycans (GAGs) and gene expressions for aggrecan, collagen II and Sox9. RESULTS: Histological and immunohistochemical analyses demonstrated hyaline-like cartilage regeneration in the transplanted sites particularly in Group 1. In contrast, tissues in Groups 2 and 3 demonstrated mainly fibrocartilage. The highest ICRS and O'Driscoll scorings was also observed in Group 1, while the lowest score was seen in Group 3. Similarly, the total GAG/total protein as well as chondrogenic gene levels were expressed in the same order, that is highest in Group 1 while the lowest in Group three. Significant differences between these 3 groups were observed (P <0.05). CONCLUSIONS: This study suggests that supplementing intra-articular injections of BM-MSCs following BMS knee surgery provides superior cartilage repair outcomes.

Hyaluronic acid with or without bone marrow derived-mesenchymal stem cells improves osteoarthritic knee changes in rat model: a preliminary report.

Despite being a complex degenerative joint disease, studies on osteoarthritis (OA) suggest that its progression can be reduced by the use of hyaluronic acid (HA) or mesenchymal stem cells (MSC). The present study thus aims to examine the effects of MSC, HA and the combination of HA-MSC in treating OA in rat model. The histological observations using O'Driscoll score indicate that it is the use of HA and MSC independently and not their combination that delays the progression of OA. In conclusion, the preliminary study suggest that the use of either HA or MSCs effectively reduces OA progression better than their combined use.