To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approaches, controllable differentiation of mesenchymal stem cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. Although bilayered scaffolds have been investigated in vitro and in vivo, no culture system is available to test BMSCs differentiation into bone and cartilage simultaneously in bilayered scaffolds. This study investigated a defined culture media, which supported osteoblast and chondrocyte differentiation depending on growth factors implemented in biomaterials. In 2-dimensional culture, BMSCs differentiated to chondrocytes when transforming growth factor-beta 3 (TGF-β3) was added to the defined media, whereas osteogenic differentiation was induced by adding bone morphogenetic protein 7 (BMP-7). BMSC differentiation to osteogenic and chondrogenic lineages was further strengthen in 3-dimensional culture. Proteoglycan formation, type II collagen, and aggrecan were upregulated in the defined media when BMSCs were mixed with fibrin gel impregnated with TGF-β3. Mineralization and the expression of osteogenic markers such as alkaline phosphatase, osteopontin, and osteoclacin were noticeable when BMSCs cultured in hydroxyapatite-tricalcium phosphate (HA/TCP) scaffolds coated with BMP-7.

This study generated and tested a growth media, which could induce osteogenic and chondrogenic differentiation of BMSCs in one culture system. These results will help the development of tissue substitutes for multi-complexed tissues such as subchondral replacement.

Bone Tissue Engineering Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia

Adult mesenchymal stem cells (MSCs) are a topic of immense research interest in the field of tissue engineering. Since, depletion of multipotent cells has been implicated in degenerative joint diseases, cell based therapies have been proposed for tissue regeneration, especially for cartilage repair. The aim of the present study is to focus on the possibility of deriving and expanding multipotential MSCs from the heterogeneous bone marrow stromal samples of patients with osteoarthritis (OA) by characterising MSCs at the single cell level.

Single cell clonal cultures were established by limiting dilution of marrow stromal cells from three OA patients. A total of 14 clones with a wide variation in their cell doubling time were isolated. The clones were grouped into fast-growing and slow-growing clones. All except one of the fast-growing clones were tripotential. However the slow-growing clones showed limited differentiation potential and morphological changes associated with cellular senescence with extended duration in culture. Flow cytometric analysis did not depict any difference in the expression of the selected putative MSC cell surface markers CD29, CD44, CD90, CD105 and CD166 between fast-growing and slow-growing clones indicating a strong need to investigate for novel cell-surface markers. Further, proteomic analysis to understand the sub-cellular processes responsible for the existence of varying sub-populations identified 11 differentially expressed proteins. These proteins were reported to be associated with cellular organization, signal transduction, energy pathways and stress related proteins. Identification of signaling pathway proteins and cell cycle related proteins, such as calmodulin and caldesmon in the clonal populations, suggest that high-throughput proteomic technologies like two dimensional liquid chromatography (2D LC) coupled with mass spectrometry (MS) may facilitate the discovery of therapeutically useful biomarkers.

This study demonstrated the existence of a fast-growing multipotential MSC population from bone marrow samples of patients with OA. Therefore, despite a supposedly smaller stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSCs.

Synthetic biodegradable polymers have been utilized increasingly in pharmaceutical, medical and biomedical engineering. Control of the interaction of living cells and biomaterials surfaces is one of the major goals in the design and development of new polymeric biomaterials in tissue engineering.

In this study, a novel amphiphilic tri-block copolymer, methoxy-terminated poly (ethylene glycol) (MPEG) – polyL-lactide (PLLA) – polylysine (PLL) was synthesized. Various molecular compositions of tri-block copolymers were prepared via optimising the parameters and characterized through Nuclear Magnetic Resonance and Gel Permeation Chromatography. The tri-block copolymer was then mixed with high molecular weight PLLA to form a flat film. The surface properties measured by X-ray Photoelectron Spectroscopy and Atomic Force Microscopy demonstrated high content of the PLL on the surface of PLLA film, which indicated self-segregation of MPEG-b-PLLA-b-PLL on PLLA surface. No cytotoxicity was detected in triblock copolymers, and compared to pure PLLA and diblock copolymers, the triblock copolymers were much more effective for cell adhesion and proliferation. It was noted that the hydrophilic chain of PEG and PLL stretched out and formed an outer layer, especially under the aqueous environment, which resulted in enhanced cell attachment and proliferation. The self-segregation behaviour of MPEG-b-PLLA-b-PLL triblock copolymer shows a potential application in scaffold preparation of tissue engineering.

Interactions between cells and polymers are mediated by proteins, which are either secreted by cells and immobilized on the biomaterial surface, or absorbed from the medium. Poly (lactic acid) (PLA) is widely used in tissue engineering as a scaffold material, however anchorage-dependent cells such as osteoblasts do not attach, grow, and differentiate well on a hydrophobic surface. In this study, a hydrophilic polymer-poly (ethylene glycol) (PEG) was used to develop diblock polymers, Methoxy-terminated poly (ethylene glycol)-Poly (lactic acid) (MPEG-PLA) to investigate cell-biomaterial interactions. Osteoblasts were cultured on different composition of PEG-PLA films in serum free or serum condition. Lactate dehydrogense (LDH) assay was used to assess the cytotoxicity of the copolymers and cell attachment and proliferation on the polymer surfaces; furthermore cell morphology was visualized by Crystal Violet stain.

The results showed that MPEG-PLA films induced early osteoblast attachment in serum free condition and the higher content of PEG in the MPEG-PLA films the more cell attachment was noticed. No significant difference of cell attachment was observed on MPEG-PLA films between serum free and 10% serum culture condition. Crystal Violet stain demonstrated the same trend in the cell-spreading characteristics on the polymer surface.

In conclusion MPEG-PLA copolymer can enhance osteoblast attachment under serum-free condition, which implies a potential application in cell delivery therapy due to the restriction in animal products for human therapeutically goods.

In both physiological and pathological processes, periosteum plays a determinant role in both bone formation and fracture healing. However, no specific reports are available so far focusing on the detailed structural and major cellular differences between the periostea covering different bone surface areas in relation to ageing. The aim of this study is to compare the structural and cellular differences in diaphyseal and epiphyseal periostea in different-aged rats using histological and immunohistochemical methods.

Four female Lewis rats from each group of juvenile (7-week old), mature (7-month old) and aged groups (2-year old) were sacrificed and the right femur of each rat was retrieved, fixed, decalcified and embedded. 5μm thick serial sagittal sections were cut and stained with Hematoxylin and Eosin, Stro-1 (stem cell marker), F4/80 (macrophage marker), TRAP (osteoclast marker) and vWF (endothelial cell marker). 1mm length of middle diaphyseal and epiphyseal periosteum were selected for observation. The thickness, total cell number and positive cell number for each antibody in each periosteal area and different-aged groups were measured and compared. The results were subjected to ANOVA and SNK-q tests.

The results showed that the thickness and cell number in diaphyseal periosteum decreased with age (p< 0.001). In comparison with diaphyseal area, the thickness and cell number in epiphyseal periosteum were much higher (p< 0.001). There were no significant differences between the juvenile and aged groups in the thickness and cell number in cambial layer of epiphyseal periosteum (p> 0.05). However, the juvenile rats had more Stro1+, F4/80+ cells and blood vessels and few TRAP+ cells in different periosteal areas compared with other groups(p< 0.001). The aged rats showed much less Stro1+ cells, but more F4/80+,TRAP+ cells and blood vessels in the cambial layer of epiphyseal periosteum (p< 0.001).

In conclusion, the age-related structure and cell population in diaphyseal and epiphyseal periostea are different, especially in aged rats. The epiphyseal periosteum of aged rats seems more destructive than diaphyseal part and other age groups. Macrophages in the periosteum play a dual important role in osteogenesis and osteoclastogenesis.

Bone Tissue Engineering Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059, Australia.

Osteophytes are the most remarkable and consistently distinct feature of osteoarthritis (OA). Their formation may be related to pluripotential cells in the periosteum responding to stimulus during OA. This study aimed to isolate stem cells from osteophyte tissues, and characterise their phenotype, proliferation and differentiation potential, and immuno-modulatory properties.

Osteophyte derived cells were isolated from five osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses.

Multipotential stem cells (MSCs) were identified from all osteophyte samples namely osteophyte derived MSCs (oMSCs). The surface antigen expression of oMSCs was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The longevity of oMSCs in culture was superior to that of bone marrow derived MSC (bMSCs), and they readily differentiated into tissues of the mesenchymal lineages. oMSCs also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO).

Our results showed that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSCs, oMSCs maintained a higher proliferative capacity, which may offer an alternative source for therapeutic stem cell based tissue regeneration.

Introduction and Aims: To deliver osteogenic cells into bone defects, the crucial steps are cell attachment and migration in cell-delivery biomaterials. The aim of this study was to examine whether complexes comprised of vitronectin (VN), insulin growth factors (IGFs) and insulin growth factor binding proteins (IGFBPs) could enhance human osteoblasts attachment, especially cell migration in three-dimensional (3-D) culture.

Method: Human osteoblasts derived from alveolar bone chips (passage 4–10) and established human osteoblast cell line SaOS-2 were used. These cells were seeded on scaffolds of type I collagen sponges and poly glycolic acid (PGA) (approx. one millimetre thick, porous structure), which had been coated with VN +/− IGF-I +/− IGFBP-3. Cell attachment and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy.

Results: The number of attached human osteoblasts was significantly higher in wells in which pre-bound VN was coated on the polystyrene culture dish or on type I collagen sponges. However, no significant difference of cell attachment was observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared to PGA scaffolds. However, coating the scaffolds with complexes comprised of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of vitronectin + IGF-I + IGFBP-5 resulted in significantly greater osteoblast migration into deep pore areas as compared to untreated scaffolds or scaffolds treated with different combinations of the VN +/− IGF +/− IGFBP-5.

Conclusion: Complexes of VN + IGFBP-5 + IGF-I enhance the attachment and migration of human osteoblast in three-dimensional culture, which implies that this complex has potential application for use in surface modification of biomaterials for tissue reconstruction.

Introduction Experimental heterotopic bone formation in the canine urinary bladder has been observed for more than seventy years without revealing the origin of the osteoinductive signals. In 1931, Huggins demonstrated bone formation in a fascial transplant to the urinary bladder. Through an elaborate set of experiments, it was found that proliferating canine transitional epithelial cells from the urinary system act as a source of osteoinduction.

Urist performed a similar series of experiments in guinea pigs as Huggins did in his canine model. After two weeks, mesenchymal cells condensed against the columnar epithelium and membranous bone with haversian systems and marrow began to form juxtapose the basement membrane. At no time was cartilage formation noted, only direct membranous bone formation. They also demonstrated the expression of BMP’s in migrating epithelium and suggested that BMP is the osteoinductive factor in heterotopic bone formation.

Method This study was approved by Institutional Animal Ethics Committee. Six dogs underwent a mid-line laparotomy incision followed by mobilisation of a right sided myoperitioneal vascularised flap based on an inferior epigastric artery pedicle. A sagittal cystotomy is made in the dome of the bladder and the vascularised flap was sutured in place with acryl absorbable, continuous suture. The animals were sacrificed at 6 weeks. The bladder samples were removed and assessed by histology and immunohistochemistry. Sections were incubated with optimal dilution of primary antibody for type I collagen, type III collagen, alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and –4, osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP).

Results The mechanism for bone formation induced by the epithelial-mesenchymal cell interactions is not clear. We were able to demonstrate mature lamellar bone formation 6 weeks after transplanting a portion of the abdominal smooth muscle into the bladder wall. The bone formed immediately adjacent to the proliferating transitional uroepithelium, a prerequisite for bone formation in Huggins’ model. Here we report evidence of cartilage formation and therefore endochondral ossification as well as membranous bone formation. This is very similar histologically to the process of endochondral ossification at the growth plate in the growing skeleton. We propose a mechanism for the expression of BMP by epithelial cells.

Discussion This study demonstrates transitional epithelium induced formation of chondrocytes and osteoblasts in muscle tissue. The sequential expression of bone matrix proteins was related to cell proliferation, differentiation and formation of endochondral and membranous bone. Further information regarding the molecular mechanism of bone formation induced by epithelial-mesenchymal cell interactions will improve understanding of cell differentiation during osteogenesis.