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Different approaches of engineering cartilage to treat defects in the articulating surfaces of the joints have been designed, which mainly use mesenchymal stem cells or autologous chondrocytes for in situ transplantation. However, these cells are poorly characterized with respect to viability, degree of differentiation and morphology or production of extracellular matrix. At present, one of the key approaches to generate chondrocytes is the stimulation of stem cells with transforming growth factor (TGF) β1. To characterize the molecular alterations occurring during the cellular transformation induced by TGF-β1 exposure, the differentiation process of bone marrow-derived stem cells into chondrocytes was investigated using an in vitro chondrogenesis model and the RNA arbitrarily primed PCR (RAP-PCR) fingerprinting technique. Distinct genes were found to be differentially regulated during chondrocyte development beginning on day 1: collagen type I, non-muscle myosin MYH9, followed by manganese superoxide dismutase and sodium-potassium ATPase on day 7. The results suggest that using RAP-PCR for differential display fingerprinting is a useful tool to investigate the differentiation process of bone marrow-derived stem cells following TGF-β1-stimulation.

Articular cartilage possesses little capacity for endogenous repair after having been damaged by disease or trauma. Various surgical procedures depending on ingrowth of mesenchymal stem cells into the defects showed repair with fibrocartilage which is of minor quality and less resistant against physical forces. New treatment options using Tissue Engineering strategies for cartilage repair showed intriguing results. Human mesenchymal stem cells (MSC) isolated from bone marrow are becoming increasingly recognized for their potential to generate different cell types and thereby function effectively in vitro or in vivo in tissue repair. Incorporation of MSCs in suitable tissue engineering scaffolds and culture in chondrogenic medium can produce cartilage-like tissue. MSCs can be harvested from bone marrow by a small puncture of the iliac crest of patients. In contrast to chondral based repair this small procedure creates no additional harvest defect in the knee joints of the patient. Numerous publications show the beneficial influence of mechanobiological conditioning (e.g. mechanical compression, hydrostatic pressure, osmotic, shear, ultrasound) on the chondrogenic differentiation of dedifferentiated chondrocytes. In contrast to chondrocytes and cartilage explants there are few studies that examine the influence of mechanobiological stress on mesenchymal progenitor cells undergoing chondrogenesis. Using an in vitro aggregate culture system enhanced chondrogenesis of mesenchymal progenitor cells, detected by an increased extracellular matrix deposition of collagen and aggrecan, could be shown under repeated cyclic hydrostatic pressure. Similar results, with an increase in chondrogenic differentiation of mesenchymal progenitor cells could be detected, when the cells were loaded in three-dimensional matrices and subjected to cyclic, compressive load or low-intensity pulsed ultrasound. This review will summarize the current state of knowledge in the field of mechanobiological conditioning of mesenchymal stem cells and its possible clinical application.

It was shown recently that the deposition of thin films of tantalum and tantalum oxide enhanced the long-term biocompatibility of stainless steel biomaterials due to an increase in their corrosion resistance. In this study, we used this tantalum oxide coating as a basis for covalent immobilization of a collagen layer, which should result in a further improvement of implant tissue integration. Because of the high degradation rate of natural collagen in vivo, covalent immobilization as well as carbodiimide induced cross-linking of the protein was performed. It was found that the combination of the silane-coupling agent aminopropyl triethoxysilane and the linker molecule N,N'-disulphosuccinimidyl suberate was a very effective system for collagen immobilizing. Mechanical and enzymatic stability testing revealed a higher stability of covalent bound collagen layers compared to physically adsorbed collagen layers. The biological response induced by the surface modifications was evaluated by in vitro cell culture with human mesenchymal stem cells as well as by in vivo subcutaneous implantation into nude mice. The presence of collagen clearly improved the cytocompatibility of the stainless steel implants which, nevertheless, significantly depended on the cross-linking degree of the collagen layer.

The effects of cyclic, mechanical compression on human bone marrow-derived mesenchymal progenitor cells undergoing chondrogenic differentiation were examined in this study. Mesenchymal progenitor cells were injected into cylindrical biodegradable scaffolds (hyaluronan-gelatin composites), cultured in a defined, serum-free chondrogenic medium and subjected to cyclic, mechanical compression. Scaffolds were loaded for 4 hours daily in the first 7 days of culture. At 1, 7, 14 and 21 days of culture, scaffolds were harvested for reverse transcriptase Polymerase Chain Reaction (RT-PCR), histology, quantitative DNA, proteoglycan and collagen analysis. Scaffolds loaded for 7 days showed a significant upregulation especially of chondrogenic markers (type II collagen, aggrecan; p<0.0001). No significant difference could be found for DNA content between loaded samples and unloaded controls. At day 1 in culture no significant differences in proteoglycan- and collagen contents could be detected between unloaded and loaded samples. After 21 days the proteoglycan (p<0.001) and collagen contents (p<0.0001) were significantly higher in the loaded samples compared to unloaded controls. By histological analysis (toluidine blue) a higher amount of proteoglycan-rich, extracellular matrix production throughout the matrix could be detected for loaded samples compared to unloaded controls. This study indicates that cyclic, mechanical compression enhances the expression of chondrogenic markers in mesenchymal progenitor cells differentiated in vitro resulting in an increased cartilaginous matrix formation, and suggests that mechanical forces may play an important role in cartilage repair.

The aim of this study was to investigate the potential of a composite matrix, containing esterified hyaluronic acid and gelatin, to facilitate the osteochondral differentiation of culture-expanded, bone marrow-derived mesenchymal progenitor cells. The cell loading characteristics and the effects of the matrix on cell differentiation were examined in vitro and in vivo. Empty and cell-loaded composites were cultivated for up to 28 days in a chemically defined medium with or without transforming growth factor-beta1 (TGF-beta1). A type II collagen-rich extracellular matrix was produced by cells loaded in the matrix and cultured in the presence of TGF-beta1. Empty and cell-loaded matrices were also implanted subcutaneously in immunodeficient mice. Three types of implant were used: empty (group I), cell-loaded matrices (Group II), and cell-loaded matrices cultured for 14 days in vitro in defined medium with TGF-beta1 (group III). No osteochondral differentiation was found in implanted empty matrices; however, the matrix supported osteochondrogenic cell differentiation in the cell-loaded implants. Preculture in vitro in a chondrogenic medium increased the percentage of osteochondral tissue found in the constructs after 3 weeks. These results indicate the potential use of this composite matrix for delivery of bone marrow-derived mesenchymal progenitor cells for the repair of chondral and osseous defects. The results also indicate that this composite matrix is useful for in vitro tissue engineering.

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

This study examined the effects of low intensity pulsed ultrasound (LIPUS) on human bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation. Aggregates of mesenchymal stem cells and mesenchymal stem cells seeded in three dimensional matrices were cultured in a defined chondrogenic medium and subjected to LIPUS for the first 7 days of culture. At 1, 7, 14 and 21 days, samples were harvested for histology, immunohistochemistry, RT-PCR, and quantitative DNA and matrix macromolecule analysis. Cell aggregates with daily treatment for 20 minutes showed no significant differences for proteoglycan and collagen content during chondrogenic differentiation. However ultrasound application for 40 minutes daily resulted in a statistically significant increase of the proteoglycan and collagen content after 21 days in culture. Aggregates treated for 20 minutes daily showed decreased expression of chondrogenic genes at all time points. In contrast, 40 minutes of daily treatment of aggregates resulted in a significant increase of chondrogenic marker genes after an initial decrease at day 7 with time in culture. Ultrasound treated cell-scaffold constructs showed a significant increase of chondrogenic marker gene expression and extracellular matrix deposition. This study indicates that LIPUS can be used to enhance the chondrogenesis of mesenchymal stem cells in cell aggregates and cell-scaffold constructs. We have found a dependency on the specific treatment parameters. We hypothesize that LIPUS can be used for an improved in vitro preparation of optimized tissue engineering implants for cartilage repair. Furthermore this non-invasive method could also be of potential use in vivo for regenerative therapy of cartilage in the future. [ABSTRACT FROM AUTHOR]