In vitro Comparison of Adipogenic Differentiation in Human Adipose-Derived Stem Cells Cultured with Collagen Gel and Platelet-Rich Fibrin.

Background: Adipose-derived stem cells (ADSCs) are the most preferred cell type, based on their phenotypic characteristics, plasticity, and favorable immunological properties for applications in soft-tissue augmentation. Hence, the present in vitro study was aimed to evaluate the adipogenic differentiation potential of human ADSCs upon culturing individually with collagen gel and platelet-rich fibrin (PRF). Materials and methods: The collected lipoaspirate was used for establishing ADSCs using enzymatic digestion method. Then, the cells were analyzed for their morphology, viability, proliferation rate, population doubling time (PDT), colony-forming ability, cell surface markers expression, and osteogenic differentiation as biological properties. Further, ADSCs were evaluated for their adipogenicity using induction media alone, and by culturing with collagen gel and PRF individually for prospective tissue augmentation. Results: ADSCs were successfully established in vitro and exhibited a fibroblast-like morphology throughout the culture period. Cells had higher viability, proliferation potential and showed their ability to form colonies. The positive expression of cell surface markers and osteogenic ability confirmed the potency of ADSCs. The ADSCs cultured on collagen gel and PRF, individually, showed higher number of differentiated adipocytes than ADSCs grown with adipogenic induction medium alone. Conclusion: The extent of lipid accumulation by ADSCs was slightly higher when cultured on collagen gel than on PRF. Additional experiments are required to confirm better suitability of scaffold materials for soft-tissue regeneration.

Influence of human teeth matrix on the cellular and biological properties of dental pulp stem cells - An in vitro study.

BACKGROUND: A major challenge in bone tissue regeneration is the use of right combination of stem cells with osteoinductive biomaterials. Hence, the present in vitro study was aimed at evaluating the effect of mineralized teeth matrix (MTM) and demineralized teeth matrix (DTM) on the selected cellular and biological characteristics of human dental pulp stem cells (DPSCs). METHODS: Established DPSCs were cultured in conditioned media (CM) of MTM and DTM and analyzed on their morphology, proliferation rate, population doubling time (PDT), viability, migration ability, ploidy and expression of cell surface markers, Further, the effect of MTM and DTM on the biocompatibility and osteogenic differentiation ability of DPSCs was evaluated. RESULTS: The DPSCs exhibited a fibroblast-like morphology with >80% viability. Cells were highly proliferative with an average PDT of 61 â€‹± â€‹12 â€‹h. A greater proliferation of DPSCs in the scratched area was observed when cultured in CM of teeth matrix compared to the cells in basal media. Moreover, no chromosomal abnormalities were induced during the culture of DPSCs. Flow cytometry analysis showed that DPSCs in basal media and CM of MTM and DTM were positive for CD29, CD44, CD73, CD90 (>70%), and negative for CD34 and CD45 (<0.1%). Alizarin red staining showed the higher deposition of mineralized nodules in DPSCs cultured with DTM compared to MTM. CONCLUSION: MTM and DTM-derived CM enhanced the proliferation and selected phenotypic markers expression with no chromosomal abnormalities in DPSCs. In addition, both matrices were biocompatible with DPSCs and increased the osteogenic differentiation through higher nodule formation.

Repair of bone defect using bone marrow cells and demineralized bone matrix supplemented with polymeric materials.

We present a novel, reverse thermo-responsive (RTR) polymeric osteogenic composite comprising demineralized bone matrix (DBM) and unmanipulated bone marrow cells (BMC) for repair of bone defects. The polymers investigated were low viscosity aqueous solutions at ambient temperature, which gel once they heat up and reach body temperature. Our goal to supplement DBM-BMC composite with RTR polymers displaying superior rheological properties, was to improve graft integrity and stability, during tissue regeneration. The osteogenic composite when implanted under kidney capsule of mice, proved to be biocompatible and biodegradable, with no residual polymer detected in the newly formed osteohematopoietic site. Implantation of the osteogenic composite into a large area of missing area of parietal bone of the skull of rats, resulted in an extensive remodeling of DBM particles, fully reconstituted hematopoietic microenvironment and well integrated normal flat bone within thirty days. The quality and shape of the newly created bone were comparable to the original bone and neither local or systemic inflammatory reactions nor fibrosis at the junction of the new and old calvarium could be documented. Furthermore, combined laser capture microdissection (LCM) technique and PCR analysis of male BMC in female rats confirmed the presence of male derived cells captured from the repaired/ regenerated flat bone defect. The use of active self sufficient osteogenic DBM-BMC composite supported by a viscous polymeric scaffold for purposive local hard tissue formation, may have a significant potential in enhancement of bone regeneration and repair following trauma, degenerative or inflamatory lesion, iatrogenic interventions and cosmetic indications.

Immunomodulation and neuroprotection with mesenchymal bone marrow stem cells (MSCs): a proposed treatment for multiple sclerosis and other neuroimmunological/neurodegenerative diseases.

Bone marrow (BM) derived mesenchymal stem cells (MSCs) (non-hematopoietic, stromal cells) can differentiate under certain circumstances into cells from various neuronal and glial type lineages; they also exert immunomodulatory effects. For potential clinical applications, BM-MSCs offer significant practical advantages over other types of stem cells, since they can be obtained from the adult BM (the patient himself being the donor) and can be easily cultured and expanded posing in parallel a very low risk for development of malignancies. We have shown that BM-MSCs cultured with a cocktail of growth factors (containing FGF and BDNF) differentiate into neuronal/glial lineage cells with a predominance of cells expressing astrocytes' markers. BM-MSCs were effective in suppression of chronic EAE in mice and induced neuroprotection, preserving most of the axons in the CNS of successfully-treated animals. Histopathological studies revealed that MSCs could efficiently migrate into the CNS inflamed tissue (both when administered intravenously and intraventricularly) and differentiated into cells expressing neural-glial lineage markers. Our preclinical results indicate that bone marrow can provide a source of stem cells with a potential for migration into inflamed CNS tissue and differentiation into cells expressing neuronal and glial cell markers. Such an approach may provide a feasible and practical way for in situ immunomodulation, neuroprotection and possibly remyelination/regeneration in diseases like multiple sclerosis. We therefore developed a explorative protocol for the evaluation of this therapeutic approach in a small group of patients with MS and other neurodegenerative diseases.

Reconstruction of cartilage, bone, and hematopoietic microenvironment with demineralized bone matrix and bone marrow cells.

Highly specialized hard tissues, such as cartilage, bone, and stromal microenvironment supporting hematopoiesis, originate from a common type of mesenchymal progenitor cell (MPC). We hypothesized that MPCs present in bone marrow cell suspension and demineralized bone matrix (DBM) that possess natural conductive and inductive features might constitute a unit containing all the essential elements for purposive bone and cartilage induction. Using a rodent preclinical model, we found that implantation of a composite comprising DBM and MPCs into A) a damaged area of a joint; B) an ablated bone marrow cavity, and C) a calvarial defect resulted in the generation of A) a new osteochondral complex comprising articular cartilage and subchondral bone; B) trabecular bone and stromal microenvironment supporting hematopoiesis, and C) flat bone, respectively. The new tissue formation followed differentiation pathways controlled by site-specific physiological conditions, thus developing tissues that precisely met local demands.

The T-box transcription factor Brachyury mediates cartilage development in mesenchymal stem cell line C3H10T1/2.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 identified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.