Both Human Hematoma Punctured from Pelvic Fractures and Serum Increase Muscle Resident Stem Cells Response to BMP9: A Multivariate Statistical Approach.

Hematoma and skeletal muscles play a crucial role in bone fracture healing. The muscle resident mesenchymal stromal cells (mrSCs) can promote bone formation by differentiating into osteoblasts upon treatment by bone morphogenetic proteins (BMP), such as BMP9. However, the influence of hematoma fracture extracts (Hema) on human mrSC (hmrSC) response to BMP9 is still unknown. We therefore determined the influence of Hema, human healthy serum (HH), and fetal bovine serum (FBS, control) on BMP9-induced osteoblast commitment of hmrSC by measuring alkaline phosphatase activity. Multiplex assays of 90 cytokines were performed to characterize HH and Hema composition and allow their classification by a multivariate statistical approach depending on their expression levels. We confirmed that BMP9 had a greater effect on osteoblastic differentiation of hmrSCs than BMP2 in presence of FBS. The hmrSCs response to BMP9 was enhanced by both Hema and HH, even though several cytokines were upregulated (IL-6, IL-8, MCP-1, VEGF-A and osteopontin), downregulated (BMP9, PDGF) or similar (TNF-alpha) in Hema compared with HH. Thus, hematoma may potentiate BMP9-induced osteogenic differentiation of hmrSCs during bone fracture healing. The multivariate statistical analyses will help to identify the cytokines involved in such phenomenon leading to normal or pathological bone healing.

Chitosan Microbeads Produced by One-Step Scalable Stirred Emulsification: A Promising Process for Cell Therapy Applications.

Cell microencapsulation is a promising approach to improve cell therapy outcomes by protecting injected cells from rapid dispersion and allowing bidirectional diffusion of nutrients, oxygen, and waste that promote cell survival in the target tissues. Here, we describe a simple and scalable emulsification method to encapsulate animal cells in chitosan microbeads using thermosensitive gel formulations without any chemical modification and cross-linker. The process consists of a water-in-oil emulsion where the aqueous phase droplets contain cells (L929 fibroblasts or human mesenchymal stromal cells), chitosan acidic solution and gelling agents (sodium hydrogen carbonate and phosphate buffer or beta-glycerophosphate). The oil temperature is maintained at 37 °C, allowing rapid physical gelation of the microbeads. Alginate beads prepared with the same method were used as a control. Microbeads with a diameter of 300-450 µm were successfully produced. Chitosan and alginate (2% w/v) microbeads presented similar rigidity in compression, but chitosan microbeads endured >80% strain without rupture, while alginate microbeads presented fragile breakage at <50% strain. High cell viability and metabolic activity were observed after up to 7 days in culture for encapsulated cells. Mesenchymal stromal cells encapsulated in chitosan microbeads released higher amounts of the vascular endothelial growth factor after 24 h compared to the cells encapsulated in manually cast macrogels. Moreover, microbeads were injectable through 23G needles without significant deformation or rupture. The emulsion-generated chitosan microbeads are a promising delivery vehicle for therapeutic cells because of their cytocompatibility, biodegradation, mechanical strength, and injectability. Clinical-scale encapsulation of therapeutic cells such as mesenchymal stromal cells in chitosan microbeads can readily be achieved using this simple and scalable emulsion-based process.

Injectable Chitosan Hydrogels with Enhanced Mechanical Properties for Nucleus Pulposus Regeneration.

IMPACT STATEMENT: A thermosensitive chitosan-based hydrogel was developed, which mimics the mechanical properties of the human nucleus pulposus (NP) tissue and provides a suitable environment for seeded NP cells to live and produce glycosaminoglycans. This scaffold is injectable through 25G needle and rapidly gels in vivo at body temperature. It has the potential to restore mechanical properties and stimulate biological repair of the degenerated intervertebral disc (IVD). It could therefore be used for the minimally invasive treatment of degenerated IVD, which affects more than one person out of five in the world.

An injectable chitosan/chondroitin sulfate hydrogel with tunable mechanical properties for cell therapy/tissue engineering.

Chitosan (CH) hydrogels with remarkable mechanical properties and rapid gelation rate were recently synthesized by combining sodium hydrogen carbonate (SHC) with another weak base, such as beta-glycerophosphate (BGP). To improve their biological responses, in the present study, chondroitin sulfate (CS) was added to these CH hydrogels. Hydrogel characteristics in terms of pH and osmolarity, as well as rheological, mechanical, morphological and swelling properties, were studied in the absence and presence of CS. Effect of CS addition on cytocompatibility of hydrogels was also assessed by evaluating the viability and metabolic activity of encapsulated L929 fibroblasts. New CH hydrogels containing CS were thermosensitive and injectable with pH and osmolality close to physiological levels and enhanced swelling capacity. Encapsulated cells were able to maintain their viability and proliferative capacity up to 7 days and CS addition improved the viability of the cells, particularly in serum-free conditions. Addition of CS showed a reducing and dose-dependent effect on the mechanical strength of the hydrogels after complete gelation. This work provides evidence that CH-CS hydrogels prepared with a combination of SHC and BGP as a gelling agent have a promising potential to be used as thermosensitive, injectable and biocompatible matrices with tunable mechanical properties for cell therapy applications.

A Longitudinal Low Dose µCT Analysis of Bone Healing in Mice: A Pilot Study.

Low dose microcomputed tomography (µCT) is a recently matured technique that enables the study of longitudinal bone healing and the testing of experimental treatments for bone repair. This imaging technique has been used for studying craniofacial repair in mice but not in an orthopedic context. This is mainly due to the size of the defects (approximately 1.0 mm) in long bone, which heal rapidly and may thus negatively impact the assessment of the effectiveness of experimental treatments. We developed a longitudinal low dose µCT scan analysis method combined with a new image segmentation and extraction software using Hounsfield unit (HU) scores to quantitatively monitor bone healing in small femoral cortical defects in live mice. We were able to reproducibly quantify bone healing longitudinally over time with three observers. We used high speed intramedullary reaming to prolong healing in order to circumvent the rapid healing typical of small defects. Bone healing prolongation combined with µCT imaging to study small bone defects in live mice thus shows potential as a promising tool for future preclinical research on bone healing.

Biocompatibility testing of single-walled carbon nanotubes on murine preosteoblasts: higher osteoblastic differentiation with BMP-9 than with BMP-2.

Carbon nanotubes (CNTs) have been used in orthopaedic applications because of their exceptional mechanical properties. However, the influence of CNTs on the behaviour of bone-forming cells and on the ability of these cells to respond to growth factors, such as bone morphogenetic proteins (BMPs), remains poorly known. Therefore, in the present study, single-walled CNTs (SWCNTs) were synthesised using an induction thermal plasma process and purified using a multistep procedure. The impact of these purified SWCNTs on the Smad activation, cell proliferation and differentiation, with or without BMP-2 and BMP-9 (1.92 nM), was also studied using western blot, mitochondrial enzymatic activity, TUNEL, RT-PCR and alkaline phosphatase activity analyses. Pre-treatment of MC3T3-E1 preosteoblasts with SWCNTs accelerated the Smad1/5/8 activation, induced by both BMP-2 and BMP-9, within 15 min. It also slightly affected their proliferation at 48 h without apoptosis. Interestingly, at 72 h, BMP-9 favoured the differentiation of MC3T3-E1 preosteoblasts pretreated with SWCNTs to a larger extent than BMP-2 did. Therefore, the combination of BMP-9 with SWCNTs appears to be a promising avenue for bone applications.

Preosteoblasts behavior in contact with single-walled carbon nanotubes synthesized by radio frequency induction thermal plasma using various catalysts.

The influence of single-walled carbon nanotubes (SWCNTs) produced by radio frequency (RF) induction thermal plasma with three catalyst mixtures (Ni-Co-Y2 O3 , Ni-Y2 O3 and Ni-Mo-Y2 O3 ) was evaluated on the behavior of murine MC3T3-E1 preosteoblasts. After analyzing SWCNTs properties, mitochondrial enzymatic (MTS) and lactate dehydrogenase (LDH) activities as well as neutral red (NR) uptake were measured to assess the cellular viability. To ascertain that the cytotoxicity was not merely as a result of the mechanical disturbance, either SWCNTs were added on the attached cells or cells were seeded on the SWCNT-covered plates. Regardless of the catalyst mixtures used for their production, SWCNTs added on the attached cells reduced cell viability drastically in a dose-dependent manner. However, the viability of cells seeded on SWCNTs even on those produced with Ni-Co-Y2 O3 was slightly decreased at 24 h and besides cells could proliferate within 48 h. Furthermore, cells were able to organize normal filamentous actin cytoskeleton and no apoptotic cells were detected in the cultures. Thus except mechanical disturbance, thermal plasma grown SWCNTs seem to induce no severe cytotoxicity on MC3T3-E1 preosteoblasts and therefore are considered promising CNTs to be studied more deeply for future applications in bone tissue engineering.

Induction thermal plasma process modifies the physicochemical properties of materials used for carbon nanotube production, influencing their cytotoxicity.

The effect of radio frequency induction thermal plasma (RFITP) process on the cytotoxicity of materials used for single-walled carbon nanotube production remains unknown. In this study, the influence of RFITP process on physicochemical and cytotoxic properties of commercial Co, Ni, Y2O3, Mo catalysts and carbon black was investigated. The cytotoxic assays (MTS, LDH, neutral red, TUNEL) revealed the strongest effect of commercial Co on murine Swiss 3T3 fibroblasts affecting their viability in a dose-dependent manner within 24 h. The cells contained also less actin stress fibres. Although RFITP affects the properties of each catalyst (size, morphology, chemistry), only cytotoxicity of Ni catalyst was increased. The plasma-treated Ni induced apoptosis. Comparing Ni particles before and after RFITP process with commercial nanoparticles of Ni revealed that the particles with similar surface area have different cytotoxicities. Interestingly, the observed toxicity of the catalysts was not mainly due to the release of ions.