In vivo cartilage repair using adipose‐derived stem cell‐loaded decellularized cartilage ECM scaffolds

We have previously reported a natural, human cartilage ECM (extracellular matrix)‐derived three‐dimensional (3D) porous acellular scaffold for in vivo cartilage tissue engineering in nude mice. However, the in vivo repair effects of this scaffold are still unknown. The aim of this study was to further explore the feasibility of application of cell‐loaded scaffolds, using autologous adipose‐derived stem cells (ADSCs), for cartilage defect repair in rabbits. A defect 4 mm in diameter was created on the patellar groove of the femur in both knees, and was repaired with the chondrogenically induced ADSC–scaffold constructs (group A) or the scaffold alone (group B); defects without treatment were used as controls (group C). The results showed that in group A all defects were fully filled with repair tissue and at 6 months post‐surgery most of the repair site was filled with hyaline cartilage. In contrast, in group B all defects were partially filled with repair tissue, but only half of the repair tissue was hyaline cartilage. Defects were only filled with fibrotic tissue in group C. Indeed, histological grading score analysis revealed that an average score in group A was higher than in groups B and C. GAG and type II collagen content and biomechanical property detection showed that the group A levels approached those of normal cartilage. In conclusion, ADSC‐loaded cartilage ECM scaffolds induced cartilage repair tissue comparable to native cartilage in terms of mechanical properties and biochemical components. Copyright © 2012 John Wiley & Sons, Ltd.     

PGA‐associated heterotopic chondrocyte cocultures: implications of nasoseptal and auricular chondrocytes in articular cartilage repair

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non‐articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell‐cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid‐ (PGA) associated porcine heterotopic mono‐ and cocultures were assessed for cell vitality, types II, I, and total collagen‐, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1‐integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species‐dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan‐rich hyaline‐like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time‐dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair. Copyright © 2011 John Wiley & Sons, Ltd.     

Efficient transfection of MG‐63 osteoblasts using magnetic nanoparticles and oscillating magnetic fields

Aims: To examine the potential of magnetic nanoparticles (MNPs) in transfecting human osteosarcoma fibroblasts (MG‐63) and investigate the effects of a novel non‐viral oscillating nanomagnetic gene transfection system (magnefect‐nano?) in enhancing transfection efficiency (TE). Methods: MG‐63 cells were transfected using MNPs coupled with a GFP‐carrying plasmid. The magnefect‐nano system was evaluated for transfection efficiency and potential associated effects on cell viability. Results: MG‐63 cells were efficiently transfected using MNPs and the magnefect‐nano system significantly enhanced overall transfection efficiency. MNPs were not found to affect cell viability and/or function of the cells. Conclusion: Non‐viral transfection using MNPs and the magnefect‐nano system can be used to transfect MG‐63 cells and assist reporter gene delivery on a single cell basis, highlighting the wide potential of nanomagnetic gene transfection in gene therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Surface modification of titanium with hydroxyapatite–heparin–BMP‐2 enhances the efficacy of bone formation and osseointegration in vitro and in vivo

Surface‐modified titanium (Ti) samples with hydroxyapatite (HAp) and heparin (Hep)–bone morphogenetic protein‐2 (BMP‐2) complex (Ti/HAp/Hep/BMP‐2) were prepared, and their efficacies on the enhancements of bone formation and osseointegration in vitro and in vivo were examined as compared to Ti/HAp and Ti/Hep/BMP‐2. The modified surfaces were characterized by X‐ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle goniometry. In vitro studies revealed that MG‐63 human osteosarcoma cell lines grown on Ti/HAp/Hep/BMP‐2 increased the amounts of alkaline phosphatase (ALP) activity, calcium deposition and the levels of OCN mRNA gene expression as compared to those grown on Ti/HAp, Ti/Hep/BMP‐2 or pristine Ti. Moreover, Ti/HAp/Hep/BMP‐2 exhibited higher bone volume (BV), bone volume/tissue volume (BV/TV), removal torque value and bone–implant contact (BIC) than Ti/HAp, Ti/Hep/BMP‐2 or pristine Ti in vivo. Histological evaluations showed that many desirable features of bone remodelling existed at the interface between Ti/HAp/Hep/BMP‐2 and the host bone. Consequently, Ti/HAp/Hep/BMP‐2 may have potential for clinical use as dental or orthopaedic implants. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell factory‐derived bioactive molecules with polymeric cryogel scaffold enhance the repair of subchondral cartilage defect in rabbits

We have explored the potential of cell factory‐derived bioactive molecules, isolated from conditioned media of primary goat chondrocytes, for the repair of subchondral cartilage defects. Enzyme‐linked immunosorbent assay (ELISA) confirms the presence of transforming growth factor‐β1 in an isolated protein fraction (12.56 ± 1.15 ng/mg protein fraction). These bioactive molecules were used alone or with chitosan–agarose–gelatin cryogel scaffolds, with and without chondrocytes, to check whether combined approaches further enhance cartilage repair. To evaluate this, an in vivo study was conducted on New Zealand rabbits in which a subchondral defect (4.5 mm wide × 4.5 mm deep) was surgically created. Starting after the operation, bioactive molecules were injected at the defect site at regular intervals of 14 days. Histopathological analysis showed that rabbits treated with bioactive molecules alone had cartilage regeneration after 4 weeks. However, rabbits treated with bioactive molecules along with scaffolds, with or without cells, showed cartilage formation after 3 weeks; 6 weeks after surgery, the cartilage regenerated in rabbits treated with either bioactive molecules alone or in combinations showed morphological similarities to native cartilage. No systemic cytotoxicity or inflammatory response was induced by any of the treatments. Further, ELISA was done to determine systemic toxicity, which showed no difference in concentration of tumour necrosis factor‐α in blood serum, before or after surgery. In conclusion, intra‐articular injection with bioactive molecules alone may be used for the repair of subchondral cartilage defects, and bioactive molecules along with chondrocyte‐seeded scaffolds further enhance the repair. Copyright © 2015 John Wiley & Sons, Ltd.