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.

Rat tail models for the assessment of injectable nucleus pulposus regeneration strategies.

Back pain is a global epidemiological and socioeconomic problem often associated with intervertebral disc degeneration; a condition believed to initiate in the nucleus pulposus (NP). There is considerable interest in developing early therapeutic interventions to target the NP and halt degeneration. Rat caudal models of disc degeneration have demonstrated significant utility in the study of disease progression and its impact on tissue structure, composition, and mechanical performance. One significant advantage of the caudal model is the ease of access and high throughput nature. However, considerable variability exists across the literature in terms of experimental setup and parameters. The objective of this article is to aid researchers in the design and development of caudal puncture models by providing details and insight into the most reported experimental parameters. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were employed to screen the existing literature and 80 manuscripts met the inclusion criteria. Disc geometry, surgical approaches, effect of needle gauge size to induce degeneration, therapeutic volume, outcome measures, and associated limitations are considered and discussed, and a range of recommendations based on different research questions are presented.

Platelet rich concentrate enhances mesenchymal stem cells capacity to repair focal cartilage injury in rabbits.

BACKGROUND: It has been previously suggested that the use of regenerative promoters, which include bone marrow-derived mesenchymal stem cells (MSCs) or natural growth factors supplement such as platelet-rich concentrate (PRC) could promote cartilage regeneration. However, the notion that the concurrent use of both promoters may provide a synergistic effect that improves the repair outcome of focal cartilage injury has not been previously demonstrated. This study was thus conducted to determine whether the concomitant use of PRC could further enhance the reparative potential of MSCs encapsulated in alginate transplanted into focal cartilage injury in rabbits. METHODS: Artifically created full thickness cartilage defects were made on the weight-bearing region of medial femoral condyles in bilateral knees of New Zealand White rabbits (N = 30). After one month, the right knee was treated with either i) PRC (n = 10), ii) MSCs (n = 10), or, iii) a combination of PRC and MSCs (PRC + MSC) (n = 10), all encapsulated in alginate. The left knee remained untreated (control). Rabbits were sacrificed at 3 and 6 months after treatment. Cartilage tissue regeneration was accessed using ICRS morphologic scoring, histologic grading by O'Driscoll scoring, immunohistochemical staining and quantitative analysis of glycosaminoglycans (GAG) per total protein content. RESULTS: At 3 months, transplantation using PRC alone was equally effective as MSCs in inducing the repair of cartilage defects. However, PRC + MSC resulted in significantly higher ICRS and O'Driscoll scores (p < 0.05) as compared to other groups. The regenerated tissues from the PRC + MSC group also had stronger staining for Safranin-O and collagen type II. By 6 months, in addition to superior ICRS and O'Driscoll scores as well as stronger staining, glycosaminoglycan per total protein content was also significantly higher (p < 0.05) in the PRC + MSC group (3.4 ±â€¯0.3 µg/mg) as compared to the MSC (2.6 ±â€¯0.2 µg/mg) or PRC (2.1 ±â€¯0.2 µg/mg) groups. CONCLUSION: PRC enhances the reparative effects of MSC in treating focal articular cartilage injuries.

Comparative efficacy of hemorrhage control of a novel mesoporous bioactive glass versus two commercial hemostats.

Mesoporous bioactive glass containing 1% Ga2O3 (1%Ga-MBG) is attractive for hemorrhage control because of its surface chemistry which can promote blood-clotting. The present study compares this proprietary inorganic coagulation accelerator with two commercial hemostats, CeloxTM (CX) and QuikClot Advanced Clotting Sponge PlusTM (ACS+). The results indicate that the number of adherent platelets were higher on the 1%Ga-MBG and CX surfaces than ACS+ whereas a greater contact activation was seen on 1%Ga-MBG and ACS+ surfaces than CX. 1%Ga-MBG not only resulted in larger platelet aggregates and more extensive platelet pseudopodia compared to CX and ACS+ but also significantly accelerated the intrinsic pathways of the clotting cascade. In vitro thrombin generation assays also showed that CX and ACS+ induced low levels of thrombin formation while 1%Ga-MBG had significantly higher values. 1%Ga-MBG formed a larger red blood cell aggregate than both CX and ACS+. Direct exposure of 1%Ga-MBG to fibroblast cells increased cell viability after 3 days relative to CX and ACS+, inferring excellent cytocompatibility. The results of this study promote 1%Ga-MBG as a promising hemostat compared to the commercially available products as it possesses essential factors required for coagulation activation.

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.

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.

Gallium-containing mesoporous bioactive glass with potent hemostatic activity and antibacterial efficacy.

Haemorrhage remains the leading cause of potentially survivable death in both military and civilian populations. Although a large variety of hemostatic agents have been developed, many of them have an inadequate capacity to induce hemostasis and are not effective in killing bacteria. In recent years, mesoporous bioactive glasses (MBGs) were found to be effective in inducing hemostasis. However, the materials may not be considered as ideal hemostats since they do not offer antimicrobial activity. The gallium ion (Ga+3) not only exhibits antibacterial properties but also accelerates the blood coagulation cascade. The aim of this study was to develop MBGs containing various concentrations of Ga2O3 (1, 2 & 3 mol%) via the evaporation-induced self-assembly (EISA) process and investigate whether the addition of Ga3+ would induce both hemostatic and antibacterial effects. The results indicated that the incorporation of lower Ga2O3 content (1 mol%) into the MBG system improved structural properties including the specific surface area, mesopore size and pore volume as well as the release of silicon and calcium ions. The bioactive glass was found to stimulate blood coagulation, platelet adhesion and thrombus generation and exerted an antibacterial effect against both Escherichia coli and Staphylococcus aureus. Likewise, Ga-doped MBGs showed excellent cytocompatibility even after 3 days, with the 1% Ga2O3-containing MBG attaining the best biocompatibility that render them safe hemostatic agents for stopping bleeding. This study demonstrated that the lowest Ga2O3-substituted MBG can be a potent candidate for controlling haemorrhage and wound infection.

Enrichment of breast cancer stem-like cells by growth on electrospun polycaprolactone-chitosan nanofiber scaffolds.

A small population of highly tumorigenic breast cancer cells has recently been identified. These cells, known as breast-cancer stem-like cells (BCSC), express markers similar to mammary stem cells, and are highly resistant to chemotherapy. Currently, study of BCSC is hampered by the inability to propagate these cells in tissue culture without inducing differentiation. Recently, it was reported that proliferation and differentiation can be modified by culturing cells on electrospun nanofibers. Here, we sought to characterize the chemoresistance and stem-like properties of breast cancer cell lines grown on nanofiber scaffolds. Cells cultured on three-dimensional templates of electrospun poly(ε-caprolactone)-chitosan nanofibers showed increases in mammary stem cell markers and in sphere-forming ability compared with cells cultured on polystyrene culture dishes. There was no increase in proliferation of stem cell populations, indicating that culture on nanofibers may inhibit differentiation of BCSC. The increase in stemness was accompanied by increases in resistance to docetaxel and doxorubicin. These data indicate that BCSC populations are enriched in cells cultured on electrospun poly(ε-caprolactone)-chitosan nanofibers, scaffolds that may provide a useful system to study BCSC and their response to anticancer drug treatment.