The effect of type I collagen on osteochondrogenic differentiation in adipose-derived stromal cells in vivo.

https://v2.sherpa.ac.uk/id/publication/27899Background The objective of this study was to investigate whether type I collagen would elicit in vivo bone formation of passaged rat adipose-derived adult stromal cells (ADASC) placed extraskeletally. Methods After expansion for 1–4 passages (P), cells were incubated in osteogenic medium containing dexamethasone, ascorbic acid and β-glycerol phosphate for 2–4 weeks. Undifferentiated cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and von Kossa staining as well as by gene expression of ALP, osteopontin (OP), osteonectin (ON), osteocalcin (OC), collagen I (colI), collagen II (colII), bone sialoprotein (BSP), periostin (Postn), runx2, osterix (Osx), sox9, msx1 and msx2. Diffusion chambers were filled with 1 × 106 cells mixed with or without type I collagen gel and implanted subcutaneously into rats. Controls included chambers exposed to (1) undifferentiated cells (with or without collagen, (2) collagen without cells and (3) empty chambers (n=5 per group). Results Four weeks after implantation, in vivo bone and cartilage formation was demonstrated in implants containing 4-week osteo-induced P1 and P4 cells wrapped in the collagen gel, as confirmed by Goldner's trichrome and Alcian blue staining, respectively. Newly formed bone stained positive for type I collagen. Control implants had no bone or cartilage and were primarily filled with fibrous tissue at that time interval. Discussion Recent studies have demonstrated that ADASC offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage and fat. However, the influence of different matrices on the in vivo osteogenic capability of ADASC is not fully understood. These findings suggest that type I collagen may support the survival and expression of osteogenic and chondrogenic phenotypes in passaged rat ADASC in vivo

Selection and induction of rat skeletal muscle-derived cells to the chondro-osteogenic lineage.

Política de acceso abierto tomada de: https://www.cellmolbiol.org/index.php/CMB/EditorialPoliciesBone marrow (BM) has been long established as the main source of pluripotential mesenchymal stem cells (MSCs), and has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The existence of an osteoprogenitor cell in other connective tissues such as skeletal muscle has been reported. In light of its availability and because of the relative ease of muscle cell isolation, skeletal muscle is an attractive source of cells for use in tissue engineering applications. The aim of this study was to explore the potential to differentiate into the chondro-osteoblastic lineage of a plastic adhering cell population, referred t as skeletal muscle-derived cells (SMDCs), obtained from biopsies of rat skeletal muscle. SMDCs displayed a fibroblast-like morphology. Our study revealed that the isolated cell population had a mesenchymal origin as indicated by abundant expression of STRO-1 and CD166. Osteogenic markers like osteocalcin (OC), bone sialoprotein (BSP)and osteopontin (OP) gene expressions were detected by RT-PCR. When these cells were cultured in the presence of an osteo-inductive culture medium, positive staining for alkaline phosphatase (ALP) and formation of mineralized matrix were increased. Furthermore SMDCs formed bone and cartilage tissues in vivo when placed inside of diffusion chambers and in demineralized bone matrix (DBM) cylinders, implanted subdermically into the backs of rat for 28 days. In conclusion, this experimental procedure is capable of selecting a cell population obtained from the skeletal muscle that is able to complete the differentiation pathway leading to the formation of cartilage and bone. In this respect SMDCs resemble BM stromal cells (BMSCs) and have demonstrated a potential application for cartilage and bone tissue engineering

A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted ubcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo

Characterization of adult stem/progenitor cell populations from bone marrow in a three-dimensional collagen gel culture system.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells that are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and nonadherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4, and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent-, hematopoietic-, mesenchymal-, and endothelial-specific markers in vitro and can undergo osteogenic differentiation in vivo

The Skeletal Atlas: A holistic approach to transcriptomics in the skeleton.

Gene expression atlases are powerful bioinformatics tools that allow access to the scientific community to explore omics data. In biomedicine, numerous gene expression databases are available, yet none provide enough information about localized transcriptomics in the skeletal system. In this work we try to solve this gap of knowledge by creating a transcriptomic atlas which brings together different works across the skeletal system, analyzing several key tissue compartments in normal and pathological conditions from mouse and human after analyzing more than 200 studies. Our atlas shows a user-friendly representation which allows the comparison of multiple genes across the different tissues of the skeleton: bone, cartilage, tendon, ligament, growth plate and skeletal muscle. As expected, our first finding was the identification of a significant heterogeneity between experiments from similar studies but from different labs, so we have created a dataset processing pipeline with inclusive/exclusive criteria of RNAseq experiments based in sequencing quality and purity of the analyzed tissues in search of a normalization protocol. We believe that this atlas will allow the homogenization of the public transcriptomic studies in the skeleton and will settle the basis to incorporate the upcoming data from single cell transcriptomics studies.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Use of Adipose-Derived Mesenchymal Stem Cells in Keratoconjunctivitis Sicca in a Canine Model

Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is an immune-mediated multifactorial disease, with high level of prevalence in humans and dogs. Our aim in this study was to investigate the therapeutic effects of allogeneic adipose-derived mesenchymal stromal cells (Ad-MSCs) implanted around the lacrimal glands in 12 dogs (24 eyes) with KCS, which is refractory to current available treatments. Schirmer tear test (STT) and ocular surface integrity were assessed at 0 (before treatment), 3, 6, and 9 months after treatment. Average STT values and all clinical signs showed a statistically significant change ( < 0.001) during the follow-up with reduction in all ocular parameters scored: ocular discharge, conjunctival hyperaemia, and corneal changes, and there were no signs of regression or worsening. Implanted cells were well tolerated and were effective reducing clinical signs of KCS with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that implantation of allogeneic Ad-MSCs around lacrimal glands has been found as an effective therapeutic alternative to treat dogs with KCS. These results could reinforce a good effective solution to be extrapolated to future studies in human

Safety and efficacy of the mesenchymal stem cell in feline eosinophilic keratitis treatment

Background: Feline eosinophilic keratitis (FEK) is a chronic keratopathy caused by a suspected immune mediated response to an unknown antigenic stimulus. The purpose of this study was to investigate the safety and therapeutic effects of allogeneic feline adipose-derived mesenchymal stromal cells (fAd-MSCs) implanted subconjunctival around the ocular surface lesion in five cats with FEK refractory to current available treatments. Results: FEK was diagnosed by clinical appearance and evidence of eosinophil and/or mast cells in corneal cytology. Each animal was treated with two applications of 2 × 106 million of fAd-MSCs 2 months apart. Ocular surface integrity was assessed before treatment and at 1, 3, 6 and 11 months after treatment. Clinical signs showed a significant change during the follow-up with resolution of the corneal and conjunctiva lesions and there were no signs of regression or worsening. Conclusions: Implanted cells were well-tolerated and effective reducing clinical signs of FEK with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that local implantation of allogeneic fAd-MSCs has been found as an effective therapeutic alternative to treat cats with FEK

A collagen-targeted biomimetic RGD peptide to promote osteogenesis

Osteogenesis is a complex, multifactorial process in which many different signals interact. The bone morphogenetic proteins (BMPs) are the most potent inducers of osteoblastic differentiation, although very high doses of BMPs in combination with collagen type I formulations have to be used for clinical applications. Although integrin-binding arginine-glycine-aspartic acid (RGD) biomimetic peptides have shown some promising abilities to promote the attachment of cells to biomaterials and to direct their differentiation, the linking of these peptides to collagen sponges usually implies chemical manipulation steps. In this study, we describe the design and characterization of a synthetic collagen-targeted RGD biomimetic (CBD-RGD) peptide formed from a collagen-binding domain derived from the von Willebrand factor and the integrin-binding RGD sequence. This peptide was demonstrated to bind to absorbable collagen type I sponges (ACSs) without performing any chemical linking, and to induce the differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells. Furthermore, in vivo experiments showed that ACSs functionalized with CBD-RGD and loaded with a subfunctional dose of BMP-2-formed ectopic bone in rats, while nonfunctionalized sponges loaded with the same amount of BMP-2 did not. These results indicate that the combination of this biomimetic peptide with the currently used collagen+BMP system might be a promising approach to improve osteogenesis and to reduce the doses of BMPs needed in clinical orthopedics.MICINN (BIO2009-13903-C01-01) MINECO (BIO2012-34960) Junta de Andalucía (P07-CVI-2781) ISCiii, RETICS, Red Española de Terapia Celular (TerCel) (RD12/0019/0032) ISCiii, CIBER-BBN (CB06/01/1015

Autologous Human-Derived Bone Marrow Cells Exposed to a Novel TGF-β1 Fusion Protein for the Treatment of Critically Sized Tibial Defect.

https://v2.sherpa.ac.uk/id/publication/12308We report the first clinical case of transplantation of autologous bone marrow-derived cells in vitro exposed to a novel recombinant human transforming growth factor (rhTGF)-β1 fusion protein bearing a collagen-binding domain (rhTGF-β1-F2), dexamethasone (DEX) and β-glycerophosphate (β-GP). When such culture-expanded cells were loaded into porous ceramic scaffolds and transplanted into the bone defect of a 69-year-old man, they differentiated into bone tissue. Marrow cells were obtained from the iliac crest and cultured in collagen gels impregnated with rhTGF-β1-F2. Cells were selected under serum-restricted conditions in rhTGF-β1-F2-containing medium for 10 days, expanded in 20% serum for 22 days and osteoinduced for 3 additional days in DEX/β-GP-supplemented medium. We found that the cell number harvested from rhTGF-β1-F2-treated cultures was significantly higher (2.3- to 3-fold) than that from untreated cultures. rhTGF-β1-F2 treatment also significantly increased alkaline phosphatase activity (2.2- to 5-fold) and osteocalcin synthesis, while calcium was only detected in rhTGF-β1-F2-treated cells. Eight weeks after transplantation, most of the scaffold pores were filled with bone and marrow tissue. When we tested the same human cells treated in vitro in a rat model using diffusion chambers, there was subsequent development of cartilage and bone following the subcutaneous transplantation of rhTGF-β1-F2-treated cells. This supports the suggestion that such cells were marrow-derived cells, with chondrogenic and osteogenic potential, whereas the untreated cells were not under the same conditions. The ability for differentiation into cartilage and bone tissues, combined with an extensive proliferation capacity, makes such a marrow-derived stem cell population valuable to induce bone regeneration at skeletal defect sites

Dendrimer surface orientation of the RGD peptide affects mesenchymal stem cell adhesion.

Mesenchymal stem cells (MSCs) are promising candidates for a range of tissue regeneration applications. Adequate scaffolds are necessary for their application in vivo, where interactions between cells and the surface material are critical. Arginine-glycine-aspartic acid tripeptides (RGD) were conjugated to polyamidoamine (PAMAM) dendrimers and used to pre-treat test surfaces. We demonstrate that pretreatment with dendrimer-presented tripeptides efficiently increases MSC adhesion to a polystyrene test surface, and that treatment effectiveness is related to how tripeptides are presented by the dendrimer to the cell. We tested both R-G-D–dendrimer and dendrimer–R-G-D arrangements and found the former to be optimal in terms of surface adhesion