Bioactive implants intended for rapid, robust, and durable bone tissue regeneration are presented. The implants are based on nanofibrous 3D-scaffolds of bioresorbable poly-ϵ-caprolactone mimicking the fibrillar architecture of bone matrix. Layer-by-layer nanoimmobilization of the growth factor BMP-2 in association with chitosan (CHI) or poly-L-lysine over the nanofibers is described. The osteogenetic potential of the scaffolds coated with layers of CHI and BMP-2 is demonstrated in vitro, and in vivo in mouse calvaria, through enhanced osteopontin gene expression and calcium phosphate biomineralization. The therapeutic strategy described here contributes to the field of regenerative medicine, as it proposes a route toward efficient repair of bone defects at reduced risk and cost level.

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Current strategies for jaw reconstruction require multiple procedures, to repair the bone defect, to offer sufficient support, and to place the tooth implant. The entire procedure can be painful and time-consuming, and the desired functional repair can be achieved only when both steps are successful. The ability to engineer combined tooth and bone constructs, which would grow in a coordinated fashion with the surrounding tissues, could potentially improve the clinical outcomes and also reduce patient suffering. A unique nanofibrous and active implant for bone-tooth unit regeneration and also the innervation of this bioengineered tooth are demonstrated. A nanofibrous polycaprolactone membrane is functionalized with neural growth factor, along with dental germ, and tooth innervation follows. Such innervation allows complete functionality and tissue homeostasis of the tooth, such as dentinal sensitivity, odontoblast function, masticatory forces, and blood flow.

BackgroundWe recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs).Methodology / Principal findingsWe applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-ß1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical.Conclusion / SignificanceWe demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications.

BACKGROUND Human platelet lysate (hPL) represents a powerful medium supplement for human mesenchymal stromal cell (hMSC) expansion. The recently published general chapters of the Pharmacopeia require the addition of a step of viral inactivation during the production process of such raw biological material used for cell‐based medicinal products. STUDY DESIGN AND METHODS The ability of gamma irradiation to inactivate viruses from a panel representative of the virus diversity was evaluated. The impact of gamma irradiation on hPL composition and efficiency as a supplement for hMSC culture was evaluated. RESULTS An efficient inactivation of all the viruses tested was demonstrated, with the minimum reduction factors obtained being superior to 4.5 log10 for human immunodeficiency virus (HIV) and hepatitis A virus (HAV) and superior to 5 log10 for bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and porcine parvovirus (PPV). The gamma irradiation did not affect the content in interesting biochemical factors for cell culture or in growth factors (GF), except to basic fibroblast GF (bFGF) whereas it highly impacted the contents in the factors involved in the coagulation cascade. Finally, gamma irradiated hPL remained as efficient as non‐irradiated hPL for the proliferation, clonogenic potential, differentiation potential, and immunosuppressive properties of hMSCs. CONCLUSION The feasibility of using gamma irradiation to efficiently inactivate viruses in hPL while maintaining its optimal efficacy as a supplement for hMSC expansion was demonstrated. Such an inactivated hPL represents a very attractive raw material for the efficient production of safe cellular therapy products.

Background

Honeycomb nanofibrous scaffolds were elaborated by electrospinning onto micro-patterned collectors either with poly(ϵ-caprolactone) (PCL) or poly(D, L-lactic acid) (PLA). The unimodal distribution of fiber diameters, observed for PLA, led to relatively flat scaffolds; on the other hand, the bimodal distribution of PCL fiber diameters significantly increased the relief of the scaffolds' patterns due to the preferential deposition of the thick fiber portions on the walls of the collector's patterns via preferential electrostatic interaction. Finally, a biological evaluation demonstrated the effect of the scaffolds' relief on the spatial organization of MG63 osteoblast-like cells. Mimicking hemi-osteons, cell gathering was observed inside PCL honeycomb nests with a size ranging from 80 to 360 µm.

Implants triggering rapid, robust and durable tissue regeneration are needed to shorten recovery times and decrease risks of postoperative complications for patients. Here, we describe active living collagen implants with highly promising bone regenerative properties. Bioactivity of the implants is obtained through the protective and stabilizing layer-by-layer immobilization of a protein growth factor in association with a polysaccharide (chitosan), within the form of nanocontainers decorating the collagen nanofibers. All components of the implants are US FDA approved. From both in vitro and in vivo evaluations, the sophisticated strategy described here should enhance, at a reduced cost, the safety and efficacy of the therapeutic implants in terms of large bone defects repair compared with current simplistic approaches based on the soaking of the implants with protein growth factor.