Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses

Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is considered potentially effective for cartilage repair and in-vitro chondrogenesis. We thus aimed to harness chondro-promotive effects of PRP and hypertrophic-suppressive effects of AC:MSC co-culture to achieve a more functional cartilage neo-tissue. We used PRP or conventional-differentiation chondrogenic media (ConvDiff) in MSC mono-cultures and AC:MSC co-cultures. We assessed gene expression of chondrogenic and hypertrophic markers using real-time RT-PCR and immunostaining. Alkaline-phosphatase activity (ALP) and calcium content of the pellets were quantified. We also measured VEGF and TNF-α secretion via ELISA. We showed PRP had higher chondrogenic potential (in mRNA and protein level) and hypertrophic-suppressive effects than Conv-Diff (mRNA level). Co-culturing reduced ALP while PRP increased calcium deposition. In all four groups, TNF-α was down-regulated compared to MSC controls, with co-cultures receiving ConvDiff media secreting the least. Meanwhile, the only group with increased VEGF secretion was PRP-mono-cultures. We observed synergistic effects for PRP and AC:MSC co-culture in enhancing chondrogenesis. Inclusion of AC reduced hypertrophic markers and angiogenic potential in PRP groups. We thus propose that combination of PRP and co-culture would favor chondrogenesis while alleviate but not totally eradicate undesired hypertrophic and pathologic responses

Study of Mir-29a expression in human adipose-derived mesenchymal stem cells treated by platelet-rich plasma

Introduction:  Mesenchymal stem cells (MSCs) have differentiation capacity to multi lineage cells, such as osteoblasts. As has been reported recently, osteogenic differentiation can be regulated by microRNAs. Although platelet-rich plasma (PRP) is used in the osteogenic differentiation process, the molecular mechanism of the effect of PRP on the induction of osteogenic differentiation by microRNAs is not well understood. We evaluated the effect of PRP on the expression of mir-29a as a key microRNA on the osteogenic differentiation process of hMSCs. Methods and Results: Mesenchymal cells were isolated from human adipose tissue and differentiated into osteoblasts. The effects of 10% PRP on bone differentiation evaluate by alkaline phosphatase activity and calcium deposition. We also evaluated gene expression of Runx2 and OPN along with the expression of mir-29a by Real-time PCR. Adipose-derived cells with differentiation potential to adipocyte and osteoblast cell lines, show significant increase in osteoblast differentiation rate, enzyme activity, mineralization upregulation of the mir-29a and gene markers when treated by 10% PRP. Conclusion: The present study showed that micro-RNAs such as mir-29a seem to play an active role in the process of bone differentiation during PRP treatment, which in turn affects the signaling pathways of mesenchymal stem cells. Determining the signaling pathways of PRP effect on osteogenic differentiation can optimize the use of this substance in the cell therapy for bone injury and fracture

Comparison between High Cell-Density Culture Systems for Chondrogenic Differentiation and Articular Cartilage Reconstruction of Human Mesenchymal Stem Cells: A Literature Review

High-cell density culture is based on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs), and because the high density ofcell and reduced oxygen tension are effective in chondrogenic differentiation. In the present paper, there will be a review about the methods ofchondrogenic differentiation of hMSCs that utilized in in vitro and in vivo chondrogenic differentiation of stem cells for treatment of osteoarthritis.There are three High-cell density culture systems; micromass, pellet culture, and alginate culture have been used to induce chondrogenicdifferentiation of hMSCs. Transplanted naive MSCs can cause problems such as heterogeneous populations. To overcome this problem, newstrategies for inducing differentiation of MSCs are needed. One possibility is a cell culture system. Collagen II and aggrecan are critical protein inchondrogenic differentiation. In all different methods, real time RT-PCR analysis demonstrates that collagen II and aggrecan mRNA are upregulated while collagen X and collagen I mRNA are down regulated. So these three high-density cell culture systems have been approved forchondrogenic differentiation. On the other hand, In micromass method, the induced-cartilage tissues are larger, more homogenous and rich incartilage specific collagen II, but collagen I, collagen X and hypertrophic chondrocyte features are markedly decreased compared to other culturesystem. Thus, the micromass culture system is the best tool for in vitro chondrogenic differentiation studies

Cytocompatibility of a conductive nanofibrous carbon nanotube/poly(L-lactic acid) composite scaffold intended for nerve tissue engineering

The purpose of this study was to fabricate a conductive aligned nanofibrous substrate and evaluate its suitability and cytocompatibility with neural cells for nerve tissue engineering purposes. In order to reach these goals, we first used electrospinning to fabricate single-walled carbon-nanotube (SWCNT) incorporated poly(L-Lactic acid) (PLLA) nanofibrous scaffolds and then assessed its cytocompatibility with olfactory ensheathing glial cells (OEC). The plasma treated scaffolds were characterized using scanning electron microscopy and water contact angle. OECs were isolated from olfactory bulb of GFP Sprague-Dawley rats and characterized using OEC specific markers via immunocytochemistry and flow cytometery. The cytocompatibility of the conductive aligned nano-featured scaffold was assessed using microscopy and MTT assay. We indicate that doping of PLLA polymer with SWCNT can augment the aligned nanosized substrate with conductivity, making it favorable for nerve tissue engineering. Our results demonstrated that SWCNT/PLLA composite scaffold promote the adhesion, growth, survival and proliferation of OEC. Regarding the ideal physical, topographical and electrical properties of the scaffold and the neurotrophic and migratory features of the OECs, we suggest this scaffold and the cell/scaffold construct as a promising platform for cell delivery to neural defects in nerve tissue engineering approaches

Comparative impact of platelet rich plasma and transforming growth factor-β on chondrogenic differentiation of human adipose derived stem cells

Introduction: Transforming growth factor-beta (TGF-β) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-β and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-β. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-β groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-β with lesser inflammatory and hypertrophic side effects

Neuroregenerative effects of olfactory ensheathing cells transplanted in a multi-layered conductive nanofibrous conduit in peripheral nerve repair in rats

Background: The purpose of this study was to evaluate the efficacy of a multi-layered conductive nanofibrous hollow conduit in combination with olfactory ensheathing cells (OEC) to promote peripheral nerve regeneration. We aimed to harness both the topographical and electrical cues of the aligned conductive nanofibrous singlewalled carbon nanotube/poly (L-lactic acid) (SWCNT/PLLA) scaffolds along with the neurotrophic features of OEC in a nerve tissue engineered approach. Results: We demonstrated that SWCNT/PLLA composite scaffolds support the adhesion, growth, survival and proliferation of OEC. Using microsurgical techniques, the tissue engineered nerve conduits were interposed into an 8 mm gap in sciatic nerve defects in rats. Functional recovery was evaluated using sciatic functional index (SFI) fortnightly after the surgery. Histological analyses including immunohistochemistry for S100 and NF markers along with toluidine blue staining (nerve thickness) and TEM imaging (myelin sheath thickness) of the sections from middle and distal parts of nerve grafts showed an increased regeneration in cell/scaffold group compared with cell-free scaffold and silicone groups. Neural regeneration in cell/scaffold group was very closely similar to autograft group, as deduced from SFI scores and histological assessments. Conclusions: Our results indicated that the tissue engineered construct made of rolled sheet of SWCNT/PLLA nanofibrous scaffolds and OEC could promote axonal outgrowth and peripheral nerve regeneration suggesting them as a promising alternative in nerve tissue engineering

Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N6-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2). Therefore, FTN2Azk variants with different Azk incorporation sites were designed and amber codon suppression was used for production. The functionalized FTN2Azk variants were efficiently produced in fed-batch like μ-bioreactor cultivations in the periplasm of E. coli displaying correct structure and antigen binding affinities comparable to those of wild-type FTN2. Our FTN2Azk variants with reactive handles for diverse conjugates enable tracking of recombinant protein in the production cell, pharmacological studies and translation into new pharmaceutical applications

Neural differentiation of mouse embryonic stem cells on conductive nanofiber scaffolds

Nerve tissue engineering requires suitable precursor cells as well as the necessary biochemical and physical cues to guide neurite extension and tissue development. An ideal scaffold for neural regeneration would be both fibrous and electrically conductive. We have contrasted the growth and neural differentiation of mouse embryonic stem cells on three different aligned nanofiber scaffolds composed of poly L: -lactic acid supplemented with either single- or multi-walled carbon-nanotubes. The addition of the nanotubes conferred conductivity to the nanofibers and promoted mESC neural differentiation as evidenced by an increased mature neuronal markers expression. We propose that the conductive scaffold could be a useful tool for the generation of neural tissue mimics in vitro and potentially as a scaffold for the repair of neural defects in vivo