Johann Clouet

An injectable vehicle for nucleus pulposus cell-based therapy

An injectable hydrogel, acting as a reservoir for cell delivery and mimicking the native environment, offers promise for nucleus pulposus (NP) repair and regeneration. Herein, the potential of a stabilised type II collagen hydrogel using poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-StarPEG) cross-linker, enriched with hyaluronic acid (HA) was investigated. The optimally stabilised type II collagen hydrogel was determined by assessing free amine groups, resistance to enzymatic degradation, gel point. The potential toxicity of the cross-linker was initially assessed against adipose-derived stem cells (ADSCs). After addition of HA (molar ratio type II collagen:HA 9:0, 9:1, 9:4.5, 9:9) within the hydrogel, the behaviour of the encapsulated NP cells was evaluated using cell proliferation assay, gene expression analysis, cell distribution and cell morphology. A significant decrease (p < 0.05) in the free amine groups of collagen was observed, confirming successful cross-linking. Gelation was independent of the concentration of 4S-StarPEG (8 min at 37 °C). The 1 mm cross-linked hydrogel yielded the most stable after enzymatic degradation (p < 0.05). No toxicity of the 4S-StarPEG was noted for the ADSCs. NP cell viability was high regardless of the concentration of HA (>80%). A cell proliferation was not seen after 14 days in its presence. At a gene expression level, HA did not influence NP cells phenotype after seven days in culture. After seven days in culture, the type I collagen mRNA expression was maintained (p > 0.05). The optimally stabilised and functionalised type II collagen/HA hydrogel system developed in this study shows promise as an injectable reservoir system for intervertebral disc regeneration.

From osteoarthritis treatments to future regenerative therapies for cartilage

Osteoarthritis (OA) is associated with cartilage degeneration and an accompanying inflammatory syndrome of the synovium in addition to alteration of the subchondral bone. The molecular and cellular events involved in OA have only partially been elucidated. This review provides a global view of the physiopathology of OA, as well as non-pharmacological and pharmacological treatments for the disorder. An update on surgical treatments and their indications is given with an orientation towards the management of OA and cartilage repair by cell-based regenerative therapies. These promising biological technologies will, potentially, play a major role in the treatment of cartilage-associated diseases.

Identification of phenotypic discriminating markers for intervertebral disc cells and articular chondrocytes

OBJECTIVE The present study was conducted to improve our knowledge of intervertebral disc (IVD) cell biology by comparing the phenotype of nucleus pulposus (NP) and annulus fibrosus (AF) cells with that of articular chondrocytes (ACs). METHODS Rabbit cells from NP and AF were isolated and their phenotype was compared with that of AC by real-time PCR analysis of type I (COL1A1), II (COL2A1) and V (COL5A1) collagens, aggrecan transcript (AGC1), matrix Gla protein (MGP) and Htra serine peptidase 1 (Htra1). RESULTS Transcript analysis indicated that despite certain similarities, IVD cells exhibit distinct COL2A1/COL1A1 and COL2A1/AGC1 ratios as compared with AC. The expression pattern of COL5A1, MGP and Htra1 makes it possible to define a phenotypic signature for NP and AF cells. CONCLUSIONS Our study shows that NP and AF cells exhibit a clearly distinguishable phenotype from that of AC. Type V collagen, MGP and HtrA1 could greatly help to discriminate among NP, AF and AC cells.

The lumbar intervertebral disc: From embryonic development to degeneration

Lumbar intervertebral discs (IVDs) are prone to degeneration upon skeletal maturity. In fact, this process could explain approximately 40% of the cases of low back pain in humans. Despite the efficiency of pain-relieving treatments, the scientific community seeks to develop innovative therapeutic approaches that might limit the use of invasive surgical procedures (e.g., spine fusion and arthroplasty). As a prerequisite to the development of these strategies, we must improve our fundamental knowledge regarding IVD pathophysiology. Recently, several studies have demonstrated that there is a singular phenotype associated with Nucleus pulposus (NP) cells, which is distinct from that of articular chondrocytes. In parallel, recent studies concerning the origin and development of NP cells, as well as their role in intervertebral tissue homeostasis, have yielded new insights into the complex mechanisms involved in disc degeneration. This review summarizes our current understanding of IVD physiology and the complex cell-mediated processes that contribute to IVD degeneration. Collectively, these recent advances could inspire the scientific community to explore new biotherapeutic strategies.

TGF-β1 and GDF5 Act Synergistically to Drive the Differentiation of Human Adipose Stromal Cells toward Nucleus Pulposus-like Cells.

Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor‐driven protocols to commit human adipose stromal cells (hASCs) toward NP‐like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor‐β1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP‐related genes characteristic of young healthy human NP cells. In addition, the engineered NP‐like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically‐inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. Stem Cells 2016;34:653–667

Intervertebral disc regeneration: a great challenge for tissue engineers.

The intervertebral disc (IVD) is a fibrocartilaginous tissue composed of a peripheral network of robust collagen fibers, termed the annulus fibrosus, which surrounds a hydrated gel-like central structure known as the nucleus pulposus (NP), and the inferior and superior cartilaginous endplates which provide the connection to the vertebral body. IVD aging inevitably leads to NP degeneration, with a decrease in cellularity and alterations in extracellular matrix (ECM) composition that ultimately affects the load-bearing capacity and mobility of the spine.

Crushed and Injected Buprenorphine Tablets: Characteristics of Princeps and Generic Solutions

Self-injection of high-dose buprenorphine is responsible for well-described complications. In 2011, we have been alerted by unusual but serious cutaneous complication among injection buprenorphine users. A prospective data collection identified 30 cases of necrotic cutaneous lesions after injection of filtered buprenorphine solution, among which 25 cases occurred following injection of buprenorphine generics. The main goal of our study was to put forward particularities that could explain the cutaneous complications, by qualitatively and quantitatively confronting particles present in Subutex and generics solutions. We used the same protocol that injected-buprenorphine users: generic or subutex tablets were crushed in sterile water and filtered through 2 filters commonly used (cotton-pad and sterifilt). Solutions were analyzed by laser granulometry, flow cytometry and scanning electron microscopy. We have highlighted the wide variation of the quantity and the size of the particles present in solution between the two drugs after cotton-pad filtration. The proportion of particles <10 µm is systematically higher in the generic solutions than with Subutex. All of the insoluble particles found in generic solutions contain silica, whereas non- organic element was to be identified in the insoluble particles of Subutex. One skin biopsy obtained from one patient who developed a necrotic lesion after intravenous injection of filtrated solution of buprenorphine generic, shows non-organic elements. Identification of particles in situ enables us to confirm the presence of silica in the biopsy. Actually the monitoring of patient receiving generic of buprenorphine must be strengthened.

Silica nanofibers as a new drug delivery system: a study of the protein–silica interactions

Drug delivery systems are proposed for the in situ controlled delivery of therapeutic molecules in the scope of tissue engineering. We propose herein silica nanofibers as carriers for the loading and release of bioactive proteins. The influence of pH, time and concentration on the amount of adsorbed proteins was studied. The interactions allowing loading were then studied by means of electron microscopy, zeta potential measurements, electron energy loss spectroscopy and attenuated total reflectance Fourier transform infrared analysis. Release profiles were determined and biological activities were enzymatically assessed. The first part of the work was carried out with lysozyme as a model protein, and then bioactive growth factors TGF-β1 and GDF-5 were used because their significance in human adipose stromal cell differentiation towards intervertebral disc nucleopulpocytes was previously assessed. It is demonstrated that protein-silica nanofiber interactions are mainly driven by hydrogen bonds and local electrostatic interactions. The present data thus provide a better understanding of the adsorption phenomenon involved, as well as a method to control protein adsorption and release. It is worth pointing out that the kinetic release of growth factors, up to 28 days, and their biological activity maintenance seem to be compatible with intervertebral disc regenerative medicine.

Intervertebral disc regeneration: From cell therapy to the development of novel bioinspired endogenous repair strategies

Low back pain (LBP), frequently associated with intervertebral disc (IVD) degeneration, is a major public health concern. LBP is currently managed by pharmacological treatments and, if unsuccessful, by invasive surgical procedures, which do not counteract the degenerative process. Considering that IVD cell depletion is critical in the degenerative process, the supplementation of IVD with reparative cells, associated or not with biomaterials, has been contemplated. Recently, the discovery of reparative stem/progenitor cells in the IVD has led to increased interest in the potential of endogenous repair strategies. Recruitment of these cells by specific signals might constitute an alternative strategy to cell transplantation. Here, we review the status of cell-based therapies for treating IVD degeneration and emphasize the current concept of endogenous repair as well as future perspectives. This review also highlights the challenges of the mobilization/differentiation of reparative progenitor cells through the delivery of biologics factors to stimulate IVD regeneration.

The transpedicular surgical approach for the development of intervertebral disc targeting regenerative strategies in an ovine model

PurposeTo investigate the suitability of the transpedicular approach (TPA) in a sheep model of IVD regenerative strategiesMethods24 IVD from four sheep were used. TPA and biopsies of the Nucleus pulposus (NP) were performed in 18 IVD (6 IVD control). Seven discographies were performed to assess the feasibility of injecting contrast agent. MRI, micro-CT scan, and histological analyses were performed and the accuracy of the TPA was evaluated. The effects on the vertebra and endplates were analyzed.Results83% of our biopsies or injections were located in the NP. Osseous fragments in IVD were observed in 50%. We observed two cases (11%) of rostral endplate fracture and five cases (27%) of breaching of the cortical pedicle and encroachment into the spinal canal. Two cases of perivertebral venous embolism and two of backflow through the canal of the TPA inside the vertebra were noted. Significant damage occurred to the bone structure of the vertebra and to the rostral endplate on which the IVD had been inserted.ConclusionsTPA induces damage to the endplates, and it may lead to neurological impairment and leakage of injected materials into the systemic circulation. These adverse effects must be fully considered before proceeding with TPA for IVD regenerative strategies.