Objectives

In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells.

Bone regenerative medicine aims at designing biomimetic biomaterials able to guide stem cells fate towards osteoblast lineage and prevent orthopaedic common pathogen adhesion. Owing to bone inorganic/organic composition, we herein report, using a versatile process based on simultaneous spray coating of interacting species, a calcium phosphate (CaP) / chitosan (CHI) / hyaluronic acid (HA) functionalized collagen membrane as a new strategy for bone regenerative medicine. Physicochemical characterizations of CaP-CHI-HA coating were performed by scanning electron microscopy, X-ray photoelectron and infrared spectroscopies and high-resolution transmission electron microscopy, revealing the formation of a thin coating mainly composed of non-stoichiometric crystalline hydroxyapatite dispersed into polymorphic organic film. Biocompatibility of CaP-CHI-HA coated membrane, evaluated after 7 days in contact with human mesenchymal stem cells (MSCs), showed spread, elongated and aligned cells. Metabolic activity and DNA quantification studies showed an increase in MSCs proliferation on coated membrane compared to uncoated membrane over the study time. Similarly, cytokines (IL-6, IL-8, osteoprotegerin) and growth factors (VEGF, bFGF) release in supernatant, as well as endothelial cells recruitment, were significantly increased in presence of CaP-CHI-HA coated membrane. Thus, CaP-CHI-HA coated membrane provides a suitable environment for MSCs to induce bone healing. Moreover, pro-inflammatory cytokines (IL-1β and TNF-α) secretion by human monocytes was significantly reduced on CaP-CHI-HA coating compared to LPS stimulation. CaP-CHI-HA coating also reduced significantly Staphylococcus aureus and Pseudomonas aeruginosa adhesion on the membrane, conferring a bacterial anti-adhesive surface. Based on our results, CaP-CHI-HA functionalized collagen membrane provides an interesting material for bone regeneration

Intra-articular infusions of adipose tissue-derived stem cells (ASCs) are a promising tool for bone regenerative medicine, thanks to their multilineage differentiating ability. One major limitation of ASCs is represented by the necessity to be isolated and expanded through in vitro culture, thus a strong interest was generated by the adipose stromal vascular fraction (SVF), the non-cultured fraction of ASCs. Besides the easiness of retrieval, handling and good availability, SVF is a heterogeneous population able to differentiate in vitro into osteoblasts, chondrocytes and adipocytes, according to the different stimuli received. We investigated and compared the bone regenerative potential of SVF and ASCs, through their ability to grow on SmartBone. ®. , a composite xenohybrid bone scaffold. SVF plated on SmartBone. ®. showed better osteoinductive capabilities than ASCs. Collagen I, osteocalcin and TGF↕ markedly stained the new tissue on SmartBone. ®. ; microCT analysis indicated a progressive increase in mineralised tissue apposition by quantification of newly formed trabeculae (3391 ± 270,5 vs 1825 ± 133,4, p± 0,001); an increased secretion of soluble factors stimulating osteoblasts, as VEGF (153,5 to 1278,1 pg/ml) and endothelin 1 (0,43 to 1,47 pg/ml), was detected over time. In conclusion, the usage of SVF, whose handling doesn't require manipulation in an in vitro culture, could definitively represent a benefit for a larger use in clinical applications. Our data strongly support an innovative idea for a bone regenerative medicine based on resorbable scaffold seeded with SVF, which will improve the precision of stem cells implant and the quality of new bone formation

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation. Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006

Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable in vitro biological efficacy for applications in bone models, regenerative medicine and infection. Indeed, HA-based coatings favor cells adhesion on printed and electrospun fibers. For antibacterial applications, the ion doped TCP coatings can reduce the bacterial growth and adhesion (E.coli and S.aureus) on electrospun matrices. To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage

Approximately 5 – 10% of all bone fractures are associated with impaired healing. It is thought that regenerative medicine has the potential to improve on existing treatments for non-union fractures, and the European market for such treatments is projected to reach £2.2 billion in 2010. The use of scaffolds for the delivery of both growth factors and human Marrow Stromal Cells (hMSCs) is thought to be a promising approach. It may be desirable to promote proliferation and chemotaxis of hMSCs at the defect site shortly after implantation, and differentiation in the longer term. This is likely to require a dual delivery system, capable of releasing multiple drugs with different release profiles. Our aim has been to develop a polymer scaffold capable of releasing bioactive molecules that are able to direct the differentiation of primary hMSCs down the osteoblastic lineage. We have examined two mutually compatible drug delivery systems: collagen coating for short term release, and polymer encapsulation for longer term release. Polymer scaffolds were manufactured and coated with Type I Collagen containing BMP-7. hMSCs from three different patient sources were exposed to the scaffolds for 14 days. The cells were then histochemically stained for Alkaline Phosphatase (ALP) and photographed. The areas of ALP staining were then normalised against the total cell count. Normalised ALP expression was increased compared to the controls for three different patients (‘110 ± 39% SE, n=6, p=0.005’, ‘540 ± 270% SE, n=6, p=0.001’, and ‘32 ± 17% SE, n=6’). Scaffolds were also manufactured either with 1,25 Vitamin D3 (another active compound) in a coating of Collagen, or encapsulated using proprietary methodologies. It was found that both treatments significantly increased normalised Alkaline Phosphatase expression within the 14d experimental period demonstrating release of the active 1,25 Vitamin D3 (’88 ± 37% SE, n=6, p=0.012’ and ‘100 ± 32% SE, n=6, p=0.012’ respectively). Our findings suggest that, subject to future testing and development, such bioactive scaffolds could form the basis for a dual drug delivery system, suitable for applications in bone regenerative medicine

Aims

Non-coding microRNA (miRNA) in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) may promote neuronal repair after spinal cord injury (SCI). In this paper we report on the effects of MSC-EV-microRNA-381 (miR-381) in a rodent model of SCI.

Aims

Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs).

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.

Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.