Introduction. It is essential to investigate the tribological maturation of tissue-engineered cartilage that is to be used in medical applications. The frictional performances of tissue engineered cartilage have been measured using flat counter surfaces such as stainless steel, glass or ceramics. However, the measured friction performances were significantly inferior to those of natural cartilage, likely because of cartilage adhesion to the counter surface. Tamura et al. reported that a poly (2- methacryloyloxyethyl phosphoryl-choline (MPC)) grafted surface shows low friction coefficient against cartilage without the adhesion to be equivalent to those for natural cartilage-on-cartilage friction. [1]. On the other hand, Yamamoto et al. reported that applying a relative sliding movement had a potential to alter the expression of tribological function of regenerated cartilage of chondrocytes. [2] In this paper, the effects of the relative sliding movement on the expression of bone marrow stromal cells (BMSC)s were investigated using the poly(MPC) grafted surface as a counter surface. Material and methods. BMSCs seeded onto fibroin sponge scaffolds were cultured by using the stirring chamber system (Figure 1), which can apply a relative tribological movement to the surface of the specimens. Three culture conditions were applied (dynamic in stirring chamber as frequency as 40 min [D1], as 40 sec [D2] and static in stirring chamber group [S]). The specimens were set into stirrer on a poly(MPC) grafted surface (MPC polymer coated surface, SANSYO). As a counter surface in friction tests, the poly(MPC) grafted surface was prepared by atom transfer radical polymerization, and the regenerated cartilage was prepared by seeding 5×10. 5. cells (BMSCs from rat bone marrow) onto fibroin sponge scaffolds (8 mm diameter and 1 mm thickness) and by 14 days culture. Results and Discussion. The friction coefficient in D1 group tended to be lower than that in S group. Similarly, D2 group tended to show lower value than S group (Figure 2). However, the value of D1 and D2 group was extraordinary high, compared to that of intact articular cartilage. The GAG amount of D1 and D2 group was significantly higher than that of S group. All of the groups showed Collagen type I and type II staining at the surface. S group showed wider staining region than D1 and D2 group. However there was no Alcian Blue staining (Figure 3). These results indicate that the stirring chamber system tended to improve the frictional performance of regenerated tissue. However this relative tribological movement has not a potential to induce effects on the differentiation of BMSCs to chondrocytes

Recently, the osteoregenerative properties of allograft have been enhanced by addition of autogenous skeletal stem cells to treat orthopaedic conditions characterised by lost bone stock. There are multiple disadvantages to allograft, and trabecular tantalum represents a potential alternative. This metal is widely used, although in applications where there is poor initial stability, or when it is used in conjunction with bone grafting, loading may need to be limited until sound integration has occurred. Strategies to speed up implant incorporation to surrounding bone are therefore required. This may improve patient outcomes, extending the clinical applications of tantalum as a substitute for allograft. Aim. To use tissue engineering strategies to enhance the reconstructive properties of tantalum, as an alternative to allograft. Methods. Human bone marrow stromal cells (5×10. 5. cells/ml) were cultured on blocks of trabecular tantalum or allograft for 28 days in basal and osteogenic media. Molecular profiling, confocal and scanning electron microscopy, as well as live/dead staining and biochemical assays were used to detail cell adherence, proliferation and phenotype. Results. Cells displayed extensive adherence and proliferation throughout trabecular tantalum. Samples cultured in osteogenic conditions showed abundant matrix production. Electron microscopy confirmed significant cellular growth through tantalum to a depth of 5mm. In contrast to cells cultured with allograft in both basal and osteogenic conditions, cell proliferation and biochemical assays showed significantly higher activity with tantalum than allograft. Furthermore, alkaline phosphatase (ALP) assay and molecular profiling confirmed no significant difference in expression of ALP, Runx-2, Col-1 and Sox-9 between cells cultured on tantalum and allograft. Conclusions. These studies demonstrate trabecular tantalum supports cell growth and osteogenic differentiation at least as well as allograft. Trabecular tantalum represents a good alternative to allograft for tissue engineering osteoregenerative strategies in the context of lost bone stock. Further mechanical testing and in vivo studies are on-going

Biomaterials used in regenerative medicine should be able to support and promote the growth and repair of natural tissues. Bioactive glasses (BGs) have a great potential for applications in bone tissue engineering [1, 2]. As it is well known BGs can bond to host bone and stimulate bone cells toward osteogenesis. Silicate BGs, e.g. 45S5 Bioglass® (composition in wt.%: 45 SiO. 2. , 6 P. 2. O. 5. , 24, 5 Na. 2. O and 24.5 CaO), exhibit positive characteristics for bone engineering applications considering that reactions on the material surface induce the release of critical concentrations of soluble Si, Ca, P and Na ions, which can lead to the up regulation of different genes in osteoblastic cells, which in turn promote rapid bone formation. BGs are also increasingly investigated for their angiogenic properties. This presentation is focused on cell behavior of osteoblast-like cells and osteoclast-like cells on BGs with varying sample geometry (including dense discs for material evaluation and coatings of highly porous Al. 2. O. 3. -scaffolds as an example of load-bearing implants). To obtain mechanically competent porous samples with trabecular architecture analogous to those of cancellous bone, in this study Al. 2. O. 3. scaffolds were fabricated by the well-known foam replication method and coated with Bioglass® by dip coating. The resulted geometry and porosity were proven by SEM and μCT. Originating from peripheral blood mononuclear cells formed multinucleated giant cells, i.e. osteoclast-like cells, after 3 weeks of stimulation with RANKL and M-CSF. Thus, the bioactive glass surface can be considered a promising material for bone healing, providing a surface for bone remodeling. Osteoblast-like cells and bone marrow stromal cells were seeded on dense bioactive glass substrates and coatings showing an initial inhibited cell attachment but later a strong osteogenic differentiation. Additionally, cell attachment and differentiation studies were carried out by staining cytoskeleton and measuring specific alkaline phosphatase activity. In this context, 45S5 bioactive glass surfaces can be considered a highly promising material for bone tissue regeneration, providing very fast kinetics for bone-like hydroxyapatite formation (mineralization). Our examinations revealed good results in vitro for cell seeding efficacy, cell attachment, viability, proliferation and cell penetration onto dense and porous Bioglass®-coated scaffolds. Recent in vivo investigations [3] have revealed also the angiogenic potential of bioactive glass both in particulate form and as 3D scaffolds confirming the high potential of BGs for bone regeneration strategies at different scales. Implant surfaces based on bioactive glasses offer new opportunities to develop these advanced biomaterials for the next generation of implantable devices and tissue scaffolds with desired tissue-implant interaction

Purpose. The biomechanical role of the meniscus in the knee joint is a function of its extracellular matrix which consists of type I collagen throughout, type II collagen in the inner meniscus region and glycosaminoglynated (GAG) proteins of which aggrecan is the most prevaleet. Meniscus reparative capacity is limited, particularly when a defect is located in the inner avascular portion, and menisectomy predisposes the joint to osteoarthritis. Using meniscus cells in tissue engineering strategies has been advocated to generate functional meniscus substitutes. However, meniscus cells, like chondrocytes of cartilage, lose their matrix-forming phenotype during culture expansion. Co-culture of chondrocytes with stem cells has been shown to result in enhanced matrix formation. We hypothesized that meniscus cells in co-culture with stem cells will result in increased matrix formation. Method. Tissue specimens were obtained after approval of the local ethical committee and informed consent. Menisci were obtained from 3 patients undergoing total knee arthroplasty; (53–84; mean age 66.6). Meniscus cells were isolated after digestion of menisci with collagenase II. Isolated meniscus cells were plated for 24–48 hr before use. Bone marrow aspirates were obtained from the iliac crest of 3 donors: 1 female (46) and 2 males (15 and 21) undergoing routine orthopaedic procedures. Plastic adherent bone marrow stromal cell populations were isolated and expanded under normal oxygen tension of 21%O2 in a-MEM growth media plus FGF-2 until passage 2. Cells were mixed at a variety of meniscus cells (Men): BMSC ratio including 5/95, 10/90 and 25/75, respectively. Mixed cells were centrifuged to form spherical pellets followed by culture in a defined serum free chondrogenic differentiation medium. Control groups were pure Men and pure BMSCs. Total cell number per pellet was 25×104. Pellets were cultured for 3 weeks under normal oxygen tension. Thereafter, pellets were processed: biochemically for GAG and DNA content, and histologically for Safranin-O staining of sulphated GAG and immunohistochemical analyses for collagen types I and II. Analysis was performed on a minimum of 2 independent pellets. Results. Relative to pure cell control pellets, co-cultured cell pellets of expanded human BMSCs and meniscus cells had more GAG matrix per DNA content. The amplitude of GAG enhancement in all co-cultures varied with donor and with the Men:BMSC ratio. However, the mean GAG enhancement was 1.8–6 fold. The GAG contents of pellets correlated with Safranin-O staining. Positive staining for collagens types I and II was increased in co-cultured cell pellets. Conclusion. Co-seeding of meniscus cells and stem cells on a suitable scaffold may aid the generation of functional grafts with improved biomechanical properties relative to those generated via expanded meniscus cells alone or stem cells alone

Numerous investigators have described osteogenic differentiation of bone marrow stromal cells obtained from both murine and human sources over the past decade. The ease of access and large available quantity of adipose tissue, however, makes Adipose-Derived Stem Cells (ADSC) a far more practical alternative for clinical applications, such as operative treatment of non-unions and regeneration of critical bone defects. Therefore, the primary goal of this research endeavor is to achieve osteogenic differentiation of ADSC. Previous work has already demonstrated that bone morphogenetic protein receptor 1A (BMP receptor 1A) signaling is required for healing critical bone defects. Based on this evidence, we used a lentiviral vector to increase expression of BMP receptor 1A by our stem cell population in order to direct their differentiation into the osteoblastic lineage. We harvested subcutaneous adipose tissue intraoperatively from consenting patients undergoing elective lipoplasty and panniculectomy procedures. The stromal vascular fraction was isolated from this tissue and further refined by passaging in selective media to yield a stable population of ADSC in primary culture. Both the identity and homogeneity of this stem cell population was confirmed using adipogenic induction media and differentiation cocktails. In addition, we subcloned an expression plasmid containing the BMP receptor 1A locus in tandem with green fluorescent protein (GFP) under the transcriptional control of a single promoter. This plasmid was packaged into a lentiviral vector to provide a reliable method of achieving both genomic integration and long-term expression of the BMP receptor 1A gene. Hence, transduction of ADSC using this vector resulted in overexpression of BMP receptor 1A by these multipotent cells. The GFP was then utilized as a reporter gene to screen and enrich the ADSC population for only those stem cells with a robust expression of BMP receptor 1A. The ADSC that overexpressed BMP receptor 1A were found to achieve osteogenic differentiation after 18 to 20 days of in vitro culture, as revealed by immunohistochemistry assays for osteocalcin. Osteogenic differentiation was further confirmed by alizarin red staining and quantitative PCR for alkaline phosphatase gene expression as a biomarker for the osteoblastic lineage. Our results demonstrate that stem cells derived from the adipose tissue of a patient represent a viable means of culturing autologous osteoblasts in vitro for future implantation at the site of critical bone defects. This method of attaining osseous regeneration is intuitively appealing, given the minimal donor site morbidity associated with removing subcutaneous fat. By transducing the ADSC with a lentiviral vector, we have also collected further evidence implicating the critical importance of signaling mediated by the BMP receptor 1A during osteogenesis. Further tissue engineering studies are now in progress to evaluate the osteogenic differentiation potential of these stem cells under hydrostatic and fluid flow shearing mechanical loads

Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.