Chondrogenic potential of human articular chondrocytes and skeletal stem cells: a comparative study

Regenerative medicine strategies have increasingly focused on skeletal stem cells (SSCs), in response to concerns such as donor site morbidity, dedifferentiation and limited lifespan associated with the use of articular chondrocytes for cartilage repair. The suitability of SSCs for cartilage regeneration, however, remains to be fully determined. This study has examined the chondrogenic potential of human STRO-1-immunoselected SSCs (STRO-1+ SSCs), in comparison to human articular chondrocytes (HACs), by utilising two bioengineering strategies, namely ‘‘scaffold-free’’ three-dimensional(3-D) pellet culture and culture using commercially available, highly porous, 3-D scaffolds with interconnected pore networks. STRO-1+ SSCs were isolated by magnetic-activated cell sorting from bone marrow samples of haematologically normal osteoarthritic individuals following routine hip replacement procedures. Chondrocytes were isolated by sequential enzymatic digestion of deep zone articular cartilage pieces dissected from femoral heads of the same individuals. After expansion in monolayer cultures, the harvested cell populations were centrifuged to form high-density 3-D pellets and also seeded in the 3-D scaffold membranes, followed by culture in serum-free chondrogenic media under static conditions for 21 and 28 days, respectively. Chondrogenic differentiation was determined by gene expression,histological and immunohistochemical analyses. Robust cartilage formation and expression of hyaline cartilage-specific markers were observed in both day-21 pellets and day-28 explants generated using HACs. In comparison, STRO-1+ SSCs demonstrated significantly lower chondrogenic differentiation potential and a tendency for hypertrophic differentiation in day-21 pellets. Culture of STRO-1+ SSCs in the 3-D scaffolds improved the expression of hyaline cartilage-specific markers in day-28 explants, however, was unable to prevent hypertrophic differentiation of the SSC population. The advantages of application of SSCs in tissue engineering are widely recognised; the results of this study, however, highlight the need for further development of cell culture protocols that may otherwise limit the application of this stem cell population in cartilage bioengineering strategies

Regulation of osteoblast development by Bcl-2-associated athanogene-1 (BAG-1)

BCL-2-associated athanogene-1 (BAG-1) is expressed by osteoblast-lineage cells; early embryonic lethality in Bag-1 null mice, however, has limited the investigation of BAG-1 function in osteoblast development. In the present study, bone morphogenetic protein-2/BMP-2-directed osteogenic differentiation of bone marrow stromal cells (BMSCs) of Bag-1+/- (heterozygous) female mice was decreased significantly. Genes crucial for osteogenic differentiation, bone matrix formation and mineralisation were expressed at significantly lower levels in cultures of Bag-1+/- BMSCs supplemented with BMP-2, while genes with roles in inhibition of BMP-2-directed osteoblastogenesis were significantly upregulated. 17-beta-estradiol (E2) enhanced responsiveness of BMSCs of wild-type and Bag-1+/- mice to BMP-2, and promoted robust BMP-2-stimulated osteogenic differentiation of BMSCs. BAG-1 can modulate cellular responses to E2 by regulating the establishment of functional estrogen receptors (ERs), crucially, via its interaction with heat shock proteins (HSC70/HSP70). Inhibition of BAG-1 binding to HSC70 by the small-molecule chemical inhibitor, Thioflavin-S, and a short peptide derived from the C-terminal BAG domain, which mediates binding with the ATPase domain of HSC70, resulted in significant downregulation of E2/ER-facilitated BMP-2-directed osteogenic differentiation of BMSCs. These studies demonstrate for the first time the significance of BAG-1-mediated protein-protein interactions, specifically, BAG-1-regulated activation of ER by HSC70, in modulation of E2-facilitated BMP-2-directed osteoblast development

Bone tissue engineering

Medical advances have led to a welcome increase in life expectancy. However, accompanying longevity introduces new challenges: increases in age-related diseases and associated reductions in quality of life. The loss of skeletal tissue that can accompany trauma, injury, disease or advancing years can result in significant morbidity and significant socio-economic cost and emphasise the need for new, more reliable skeletal regeneration strategies. To address the unmet need for bone augmentation, tissue engineering and regenerative medicine have come to the fore in recent years with new approaches for de novo skeletal tissue formation. Typically, these approaches seek to harness stem cells, innovative scaffolds and biological factors that promise enhanced and more reliable bone formation strategies to improve the quality of life for many. This review provides an overview of recent developments in bone tissue engineering focusing on skeletal stem cells, vascular development, bone formation and the translation from preclinical in vivo models to clinical delivery

Nanofibrous poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) scaffolds provide a functional microenvironment for cartilage repair

Articular cartilage defects, when repaired ineffectively, often lead to further deterioration of the tissue, secondary osteoarthritis and, ultimately, joint replacement. Unfortunately, current surgical procedures are unable to restore normal cartilage function. Tissue engineering of cartilage provides promising strategies for the regeneration of damaged articular cartilage. As yet, there are still significant challenges that need to be overcome to match the long-term mechanical stability and durability of native cartilage. Using electrospinning of different blends of biodegradable poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate), we produced polymer scaffolds and optimised their structure, stiffness, degradation rates and biocompatibility. Scaffolds with a poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) ratio of 1:0.25 exhibit randomly oriented fibres that closely mimic the collagen fibrillar meshwork of native cartilage and match the stiffness of native articular cartilage. Degradation of the scaffolds into products that could be easily removed from the body was indicated by changes in fibre structure, loss of molecular weight and a decrease in scaffold stiffness after one and four months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes revealed a hyaline-like cartilage matrix. The ability to fine tune the ultrastructure and mechanical properties using different blends of poly(3-hydroxybutyrate)/poly(3-hydroxyoctanoate) allows to produce a cartilage repair kit for clinical use to reduce the risk of developing secondary osteoarthritis. We further suggest the development of a toolbox with tailor-made scaffolds for the repair of other tissues that require a ‘guiding’ structure to support the body’s self-healing process

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects

BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation. <br/

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects

Stem cell-based therapies for musculoskeletal regeneration: role of the microenvironment in augmenting regenerative medicine therapies by elucidating stem cell physiology

All the information required to define a multicellular organism resides in asingle cell, the zygote or the single-cell embryo. And therein lies theorigins of the concept of a “stem cell” as a highly specialised cellcontaining all the information required to generate a complex multicellularorganism. Stem cells are a key component of the multidisciplinary tissueengineering paradigm used for the generation of living tissues and organsex vivo (outside the body). The bioengineered tissues and organs areimplanted in vivo to improve or restore normal biological function inregenerative medicine therapies for disorders of complex organ systemssuch as the musculoskeletal system. A wide array of regenerativemedicine strategies, ranging from stem cell-based therapies to theapplication of tissue-engineered products, have been applied for thetreatment of bone defects, articular cartilage lesions, disorders of thespine and tendon/ligament injuries. Further advances in the developmentof improved musculoskeletal regenerative medicine therapies will beguided by a detailed understanding of underlying mechanisms governingthe homeostasis between stem cell renewal and differentiation

Osteoblast stimulating factor (OSF-1) A peptide with diverse roles in bone development

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