A new Ag-nanostructured hydroxyapatite porous scaffold: Antibacterial effect and cytotoxicity study.

We report a new chemical method for the functionalization of Mg-hydroxyapatite (Mg-HA) scaffold with Ag nanoparticles (Ag NPs) integrating in one step both the synthesis of the Ag NPs and their nano-structuring into the HA matrix (Ag-Mg-HA scaffold). This method exploits a green photochemical synthesis and allows the direct growth of Ag NPs on the Mg-HA surface. The surface structure of Ag-Mg-HA scaffold, investigated by scanning electron microscopy, shows no significant changes in the morphology upon Ag NPs incorporation. The presence of Ag was confirmed by EDX analysis. TEM and spectroscopic investigations show Ag NPs spherical shaped with a mean diameter of about 20 nm exhibiting the typical plasmon absorption band with maximum at 420 nm. The antibacterial properties of Ag-Mg-HA scaffolds were tested against two bacterial strains, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The results show excellent antibacterial properties achieving up to 99% and 100% reduction of colonies for both bacteria cultures after 24 h of incubation and 100% of reduction after 48 h of incubation. The cytotoxicity of Ag-Mg-HA was also in deep investigated assessing both cell proliferation and differentiation using hADSCs (human Adipose Derived Stem Cells) and testing data point at 0, 7, 14 and 24 days. The results show cytotoxic effect with cell proliferation decreasing up to 90% at 24 days and osteogenic differentiation inhibition. The observed cytotoxicity can be probable ascribed to the oxidative stress by ROS. Indeed, considering the effectiveness of the nanofunctionalization method and the excellent antibacterial properties showed by the Ag-Mg-HA scaffold, future works will be devoted to create nanofunctionalized scaffold satisfying both antimicrobial and osteo-regenerative properties.

Au, Pd and maghemite nanofunctionalized hydroxyapatite scaffolds for bone regeneration.

Nanotechnology plays a key role in the development of innovative scaffolds for bone tissue engineering (BTE) allowing the incorporation of nanomaterials able to improve cell proliferation and differentiation. In this study, Mg-HA-Coll type I scaffolds (Mg-HA-based scaffolds) were nanofunctionalized with gold nanorods (Au NRs), palladium nanoparticles (Pd NPs) and maghemite nanoparticles (MAG NPs). Nanofunctionalized Mg-HA-based scaffolds (NF-HA-Ss) were tested for their ability to promote both the proliferation and the differentiation of adipose-derived mesenchymal stem cells (hADSCs). Results clearly highlight that MAG nanofunctionalization substantially improves cell proliferation up to 70% compared with the control (Mg-HA-based scaffold), whereas both Au NRs and Pd NPs nanofunctionalization induce a cell growth inhibition of 94% and 89%, respectively. Similar evidences were found for the osteoinductive properties showing relevant calcium deposits (25% higher than the control) for MAG nanofunctionalization, while a decreasing of cell differentiation (20% lower than the control) for both Au NRs and Pd NPs derivatization. These results are in agreement with previous studies that found cytotoxic effects for both Pd NPs and Au NRs. The excellent improvement of both osteoconductivity and osteoinductivity of the MAG NF-HA-S could be attributed to the high intrinsic magnetic field of superparamagnetic MAG NPs. These findings may pave the way for the development of innovative nanostructured scaffolds for BTE.

Evaluation of a Cell-Free Collagen Type I-Based Scaffold for Articular Cartilage Regeneration in an Orthotopic Rat Model.

The management of chondral defects represents a big challenge because of the limited self-healing capacity of cartilage. Many approaches in this field obtained partial satisfactory results. Cartilage tissue engineering, combining innovative scaffolds and stem cells from different sources, emerges as a promising strategy for cartilage regeneration. The aim of this study was to evaluate the capability of a cell-free collagen I-based scaffold to promote cartilaginous repair after orthotopic implantation in vivo. Articular cartilage lesions (ACL) were created at the femoropatellar groove in rat knees and cell free collagen I-based scaffolds (S) were then implanted into right knee defect for the ACL-S group. No scaffold was implanted for the ACL group. At 4-, 8- and 16-weeks post-transplantation, degrees of cartilage repair were evaluated by morphological, histochemical and gene expression analyses. Histological analysis shows the formation of fibrous tissue, at 4-weeks replaced by a tissue resembling the calcified one at 16-weeks in the ACL group. In the ACL-S group, progressive replacement of the scaffold with the newly formed cartilage-like tissue is shown, as confirmed by Alcian Blue staining. Immunohistochemical and quantitative real-time PCR (qRT-PCR) analyses display the expression of typical cartilage markers, such as collagen type I and II (ColI and ColII), Aggrecan and Sox9. The results of this study display that the collagen I-based scaffold is highly biocompatible and able to recruit host cells from the surrounding joint tissues to promote cartilaginous repair of articular defects, suggesting its use as a potential approach for cartilage tissue regeneration.

In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold.

Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i) is biocompatible in vivo, (ii) is able to recruit host cells (iii) induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse.

Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

Bone augmentation after ectopic implantation of a cell-free collagen-hydroxyapatite scaffold in the mouse.

The bone grafting is the classical way to treat large bone defects. Among the available techniques, autologous bone grafting is still the most used but, however, it can cause complications such as infection and donor site morbidity. Alternative and innovative methods rely on the development of biomaterials mimicking the structure and properties of natural bone. In this study, we characterized a cell-free scaffold, which was subcutaneously implanted in mice and then analyzed both in vivo and ex vivo after 1, 2, 4, 8 and 16 weeks, respectively. Two types of biomaterials, made of either collagen alone or collagen plus magnesium-enriched hydroxyapatite have been used. The results indicate that bone augmentation and angiogenesis could spontaneously occur into the biomaterial, probably by the recruitment of host cells, and that the composition of the scaffolds is crucial. In particular, the biomaterial more closely mimicking the native bone drives the process of bone augmentation more efficiently. Gene expression analysis and immunohistochemistry demonstrate the expression of typical markers of osteogenesis by the host cells populating the scaffold. Our data suggest that this biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.