In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

Background: Bone grafts are required to repair large bone defects after tumour resection or large tr

Trophic Effects of Mesenchymal Stem Cells in Tissue Regeneration

Mesenchymal stem cells (MSCs) are considered to hold great therapeutic value for cell-based therapy and for tissue regeneration in particular. Recent evidence indicates that the main underlying mechanism for MSCs' beneficial effects in tissue regeneration is based on their capability to produce a large variety of bioactive trophic factors that stimulate neighboring parenchymal cells to start repairing damaged tissues. These new findings could potentially replace the classical paradigm of MSC differentiation and cell replacement. These bioactive factors have diverse actions like modulating the local immune system, enhancing angiogenesis, preventing cell apoptosis, and stimulating survival, proliferation, and differentiation of resident tissue specific cells. Therefore, MSCs are referred to as conductors of tissue repair and regeneration by secreting trophic mediators. In this review article, we have summarized the studies that focused on the trophic effects of MSC within the context of tissue regeneration. We will also highlight the various underlying mechanisms used by MSCs to act as trophic mediators. Besides the secretion of growth factors, we discuss two additional mechanisms that are likely to mediate MSC's beneficial effects in tissue regeneration, namely the production of extracellular vesicles and the formation of membrane nanotubes, which can both connect different cells and transfer a variety of trophic factors varying from proteins to mRNAs and miRNAs. Furthermore, we postulate that apoptosis of the MSCs is an integral part of the trophic effect during tissue repair

Injectable Cell-Laden Polysaccharide Hydrogels: In Vivo Evaluation of Cartilage Regeneration

Previously, 5% w/v hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex-TA) enzymatically cross-linked hybrid hydrogels were demonstrated to provide a mechanically stable environment, maintain cell viability, and promote cartilaginous-specific matrix deposition in vitro. In this study, 5% w/v hybrid hydrogels were combined with human mesenchymal stem cells (hMSCs), bovine chondrocytes (bCHs), or a combination of both in a 4:1 ratio and subcutaneously implanted in the backs of male and female nude rats to assess the performance of cell-laden hydrogels in tissue formation. Subcutaneous implantation of these biomaterials showed signs of integration of the gels within the host tissue. Histological analysis showed residual fibrotic capsules four weeks after implantation. However, enhanced tissue invasion and some giant cell infiltration were observed in the HA-TA/Dex-TA hydrogels laden with either hMSCs or bCHs but not with the co-culture. Moreover, hMSC-bCH co-cultures showed beneficial interaction with the hydrogels, for instance, in enhanced cell proliferation and matrix deposition. In addition, we provide evidence that host gender has an impact on the performance of bCHs encapsulated in HA-TA/Dex-TA hydrogels. This study revealed that hydrogels laden with different types of cells result in distinct host responses. It can be concluded that 5% w/v hydrogels with a higher concentration of Dex-TA (≥50%) laden with bCH-hMSC co-cultures are adequate for injectable applications and in situ cell delivery in cartilage regeneration approaches

Nanofracturing: a new technique for bone marrow stimulation in equine cartilage repair

Microfracture is the current standard in treatment of focal full-thickness cartilage lesions in horses, but clinical outcome may vary. Nanofracture is a novel technique that uses a commercially developed device to yield smaller diameter perforations with deeper penetration into the subchondral bone. Experimentally, in rabbits and sheep, nanofracture has been shown to result in superior repair compared to microfracture. The objective was to study the feasibility and preliminary outcome of nanofracture using a commercial device for treatment of cartilage defects in horses. Nanofracture was tested ex vivo in n=2 cadaveric equine stifle joints and in vivo in n=8 horses with experimental partial thickness cartilage defects in the medial femoral trochlear ridge. These were treated with an experimental biomaterial or nanofracture, and repair tissue was studied macroscopically (ICRS-I score) and microscopically (histological ICRS-II score and micro-CT) after 7 months. Both in cadaveric equine stifle joints and in vivo, the nanofracture device could readily be applied and allowed easy penetration of the subchondral bone. Repair tissue after 7 months was graded ‘near-normal’ macroscopically, while histologically, the abundant repair tissue proved mainly fibrocartilaginous in nature. Micro-CT revealed near-full restoration of mid-lesion cartilage layer thickness but altered subchondral bone microarchitecture. The in vivo study did not include a control group treated with conventional microfracture for comparison. To our knowledge, this is the first report on bone marrow stimulation using nanofracture as a potential method to enhance chondral defect repair in horses. In the in vivo study, no clinical adverse effects were observed, and promising good defect filling with fibrocartilaginous tissue was seen 7 months after treatment.La microfractura es el estándar actual en el tratamiento de lesiones focales de cartílago de grosor completo en caballos, pero el resultado clínico puede variar. La nanofractura es una técnica novedosa que utiliza un dispositivo desarrollado comercialmente para producir perforaciones de menor diámetro con una penetración más profunda en el hueso subcondral. Experimentalmente, en conejos y ovejas, se ha demostrado que la nanofractura da lugar a una reparación superior en comparación con la microfractura. El objetivo era estudiar la viabilidad y el resultado preliminar de la nanofractura utilizando un dispositivo comercial para el tratamiento de defectos del cartílago en caballos. La nanofractura se probó ex vivo en n=2 articulaciones de la ahogada cadavérica equina e in vivo en n=8 caballos con defectos de cartílago de espesor parcial experimental en la cresta troclear del fémur medial. Estos fueron tratados con un biomaterial experimental o nanofractura, y el tejido de reparación fue estudiado macroscópicamente (puntuación ICRS-I) y microscópicamente (puntuación histológica ICRS-II y micro-TC) después de 7 meses. Tanto en las articulaciones de la ahogada equina cadavérica como in vivo, el dispositivo de nanofractura podía aplicarse fácilmente y permitía una fácil penetración del hueso ubcondral. El tejido de reparación después de 7 meses fue calificado acroscópicamente como 'casi normal', mientras que histológicamente, el abundante tejido de reparación demostró ser principalmente de naturaleza fibrocartilaginosa. La micro-TC reveló una restauración casi completa del grosor de la capa de cartílago de la lesión media, pero alteró la microarquitectura del hueso subcondral. El estudio in vivo no incluyó un grupo de control tratado con microfractura convencional para la comparación. Hasta donde sabemos, este es el primer informe sobre la estimulación de la médula ósea usando la nanofractura como un método potencial para mejorar la reparación de defectos condrales en caballos. En el estudio in vivo, no se observaron efectos clínicos adversos, y se vio un buen relleno del defecto con tejido fibrocartilaginoso 7 meses después del tratamiento.Escuela de Medicina Veterinari

A Rapid and Efficient Method for Expansion of Human Mesenchymal Stem Cells

During the past decade, there has been much interest in the use of human mesenchymal stem cells (hMSCs) in bone tissue engineering. HMSCs can be obtained relatively easily and expanded rapidly in culture, but for clinical purposes large numbers are often needed and the cost should be kept to a minimum. A rapid and efficient culturing protocol would therefore be beneficial. In this study, we examined the effect of different medium compositions on the expansion and osteogenic differentiation of bone marrow–derived hMSCs from 19 donors. We also investigated the effect of low seeding density and dexamethasone on both hMSCs expansion and their in vitro and in vivo osteogenic differentiation capacity. HMSCs seeded at a density of 100 cells/cm2 had a significantly higher growth rate than at 5000 cell/cm2, which was further improved by the addition of dexamethasone. Expanded hMSCs were characterized in vitro on the basis of positive staining for CD29, CD44, CD105, and CD166. The in vitro osteogenic potential of expanded hMSCs was assessed by flow cytometric staining for alkaline phosphatase. In vivo bone-forming potential of the hMSCs was assessed by seeding the cells in ceramic scaffolds, followed by subcutaneous implantation in nude mice and histopathologic assessment of de novo bone formation after 6-week implantation. Expanded hMSCs from all donors displayed similar osteogenic potential independent of the culture conditions. On the basis of these results we have developed an efficient method to culture hMSCs by seeding the cells at 100 cells/cm2 in an α-minimal essential medium–based medium containing dexamethasone