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Introduction: Osteoarthritis (OA), the most common form of joint disease, affects more than 500 million people worldwide. This painful and debilitating disease imposes a huge socioeconomic cost worldwide. Despite years of promising research, no etiological drug has been successfully introduced into daily clinical practice. In this context, gene therapy (GT) is emerging as a tool capable of meeting an increasingly specialized medical need. Five GT drugs for OA are currently under clinical evaluation, demonstrating the relevance of this tool. However, the widespread use of GT is still limited by considerations of safety, long-term efficacy, controlled and specific targeting, and the presence of neutralizing immune responses. Cartilage, a tissue of interest to target in OA, is a complex tissue to penetrates with the various GT vectors. Methods: This manuscript reviews current clinical trials involving DNA-based GT for OA and suggests ways to improve recombinant adenoviral and adeno-associated viral vectors, including capsid engineering and transgene sequence optimization to achieve long-term expression of a given transgene exclusively in the target joint tissue, including cartilage. Results: This review then highlights that the use of hybrid serotypes and/or chemical modifications of capsids are promising for improved tissue targeting. In addition, the choice of promoter and type of vectorized nucleic acid (single- or double-stranded DNA) appears to be critical for efficient transgene expression. Conclusion: Finally, the combination of increasing knowledge about biocompatible materials and viral vectors should also be a way to improve transduction efficiency, increase the stability of transgene expression, and allow escape from neutralizing antibodies.

The cellular microenvironment plays a major role in the biological functions of cells. Thus, biomaterials, especially hydrogels, which can be design to mimic the cellular microenvironment, are being increasingly used for cell encapsulation, delivery, and 3D culture, with the hope of controlling cell functions. Yet, much remains to be understood about the effects of cell-material interactions, and advanced synthetic strategies need to be developed to independently control the mechanical and biochemical properties of hydrogels. To address this challenge, we designed a new hyaluronic acid (HA)-based hydrogel platform using a click and bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. This approach facilitates the synthesis of hydrogels that are easy to synthesize and sterilize, have minimal swelling, are stable long term, and are cytocompatible. It provides bioorthogonal HA gels over an uncommonly large range of stiffness (0.5–45 kPa), all forming within 1–15 min. More importantly, our approach offers a versatile one-pot procedure to independently tune the hydrogel composition (e.g., polymer and adhesive peptides). Using this platform, we investigate the independent effects of polymer type, stiffness, and adhesion on the secretory properties of human adipose-derived stromal cells (hASCs) and demonstrate that HA can enhance the secretion of immunomodulatory factors by hASCs.

Summary: Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-β inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research.

Abstract Rheumatoid arthritis (RA) and periodontitis are chronic inflammatory diseases leading to increased bone resorption. Preventing this inflammatory bone resorption is a major health challenge. Both diseases share immunopathogenic similarities and a common inflammatory environment. The autoimmune response or periodontal infection stimulates certain immune actors, leading in both cases to chronic inflammation that perpetuates bone resorption. Moreover, RA and periodontitis have a strong epidemiological association that could be explained by periodontal microbial dysbiosis. This dysbiosis is believed to be involved in the initiation of RA via three mechanisms. (i) The dissemination of periodontal pathogens triggers systemic inflammation. (ii) Periodontal pathogens can induce the generation of citrullinated neoepitopes, leading to the generation of anti-citrullinated peptide autoantibodies. (iii) Intracellular danger-associated molecular patterns accelerate local and systemic inflammation. Therefore, periodontal dysbiosis could promote or sustain bone resorption in distant inflamed joints. Interestingly, in inflammatory conditions, the existence of osteoclasts distinct from “classical osteoclasts” has recently been reported. They have proinflammatory origins and functions. Several populations of osteoclast precursors have been described in RA, such as classical monocytes, a dendritic cell subtype, and arthritis-associated osteoclastogenic macrophages. The aim of this review is to synthesize knowledge on osteoclasts and their precursors in inflammatory conditions, especially in RA and periodontitis. Special attention will be given to recent data related to RA that could be of potential value in periodontitis due to the immunopathogenic similarities between the two diseases. Improving our understanding of these pathogenic mechanisms should lead to the identification of new therapeutic targets involved in the pathological inflammatory bone resorption associated with these diseases.

Abstract Bioprinting is a booming technology, with numerous applications in tissue engineering and regenerative medicine. However, most biomaterials designed for bioprinting depend on the use of sacrificial baths and/or non‐physiological stimuli. Printable biomaterials also often lack tunability in terms of their composition and mechanical properties. To address these challenges, the authors introduce a new biomaterial concept that they have termed “clickable dynamic bioinks”. These bioinks use dynamic hydrogels that can be printed, as well as chemically modified via click reactions to fine‐tune the physical and biochemical properties of printed objects after printing. Specifically, using hyaluronic acid (HA) as a polymer of interest, the authors investigate the use of a boronate ester‐based crosslinking reaction to produce dynamic hydrogels that are printable and cytocompatible, allowing for bioprinting. The resulting dynamic bioinks are chemically modified with bioorthogonal click moieties to allow for a variety of post‐printing modifications with molecules carrying the complementary click function. As proofs of concept, the authors perform various post‐printing modifications, including adjusting polymer composition (e.g., HA, chondroitin sulfate, and gelatin) and stiffness, and promoting cell adhesion via adhesive peptide immobilization (i.e., RGD peptide). The results also demonstrate that these modifications can be controlled over time and space, paving the way for 4D bioprinting applications.

Abstract An easy, reliable, and time-efficient standardized approach for assessing lumbar intervertebral disc (IVD) degeneration with relaxation times measurements in pre-clinical and clinical studies is lacking. This prospective study aims to determine the most appropriate method for lumbar IVD degeneration (IDD) assessment in sheep by comparing three quantitative MRI sequences (variable-flip-angle T1 mapping, and multi-echo T2 and T2* mapping), correlating them with Pfirrmann grading and histology. Strong intra- and interrater agreements were found for Nucleus pulposus (NP) regions-of-interest (ROI). T1, T2, and T2* mapping correlated with Pfirrmann grading and histological scoring (p

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.

Abstract In skeletal surgical procedures, bone regeneration in irregular and hard-to-reach areas may present clinical challenges. In order to overcome the limitations of traditional autologous bone grafts and bone substitutes, an extrudable and easy-to-handle innovative partially demineralized allogenic bone graft in the form of a paste has been developed. In this study, the regenerative potential of this paste was assessed and compared to its clinically used precursor form allogenic bone particles. Compared to the particular bone graft, the bone paste allowed better attachment of human mesenchymal stromal cells and their commitment towards the osteoblastic lineage, and it induced a pro-regenerative phenotype of human monocytes/macrophages. The bone paste also supported bone healing in vivo in a guide bone regeneration model and, more interestingly, exhibited a substantial bone-forming ability when implanted in a critical-size defect model in rat calvaria. Thus, these findings indicate that this novel partially demineralized allogeneic bone paste that combines substantial bone healing properties and rapid and ease-of-use may be a promising alternative to allogeneic bone grafts for bone regeneration in several clinical contexts of oral and maxillofacial bone grafting.

Abstract The osteochondral (OC) unit plays a pivotal role in joint lubrication and in the transmission of constraints to bones during movement. The OC unit does not spontaneously heal; therefore, OC defects are considered to be one of the major risk factors for developing long‐term degenerative joint diseases such as osteoarthritis. Yet, there is currently no curative treatment for OC defects, and OC regeneration remains an unmet medical challenge. In this context, a plethora of tissue engineering strategies have been envisioned over the last two decades, such as combining cells, biological molecules, and/or biomaterials, yet with little evidence of successful clinical transfer to date. This striking observation must be put into perspective with the difficulty in comparing studies to identify overall key elements for success. This systematic review aims to provide a deeper insight into the field of material‐assisted strategies for OC regeneration, with particular considerations for the therapeutic potential of the different approaches (with or without cells or biological molecules), and current OC regeneration evaluation methods. After a brief description of the biological complexity of the OC unit, the recent literature is thoroughly analyzed, and the major pitfalls, emerging key elements, and new paths to success are identified and discussed.

Background. Mesenchymal stem/stromal cells (MSCs) have been widely used for their therapeutic properties in many clinical applications including osteoarthritis. Despite promising preclinical results showing the ability of MSC to reduce the clinical severity of osteoarthritis (OA) in experimental animal models, the benefits of intra-articular injection of MSC in OA patients are limited to the short term. In this regard, it is anticipated that improving the properties of MSC may collectively enhance their long-term beneficial effects on OA. Methods and Results. Recently, we have shown that PPARβ/δ inhibition using a commercially available antagonist in murine MSC increases their immunoregulatory potential in vitro as well as their therapeutic potential in an experimental murine arthritis model. Here, we relied on an innovative strategy to inhibit PPARβ/δ:NF-κB TF65 subunit interaction in human MSC by designing and synthesizing an interfering peptide, referred to PP11. Through RT-qPCR experiments, we evidenced that the newly synthesized PP11 peptide reduced the expression level of PDK4, a PPARβ/δ target gene, but did not modify the expression levels of ACOX1 and CPT1A, PPARα target genes, and FABP4, a PPARγ target gene compared with untreated human MSC. Moreover, we showed that human MSCs pretreated with PP11 exhibit a significantly higher capacity to inhibit the proliferation of activated PBMC and to decrease the frequency of M1-like macrophages. Conclusions. We designed and synthesized an interfering peptide that potently and specifically blocks PPARβ/δ activity with concomitant enhancement of MSC immunoregulatory properties.

Autologous bone grafts (BGs) remain the reference grafting technique in various clinical contexts of bone grafting procedures despite their numerous peri- and post-operative limitations. The use of allogeneic bone is a viable option for overcoming these limitations, as it is reliable and it has been widely utilized in various forms for decades. However, the lack of versatility of conventional allogeneic BGs (e.g., blocks, powders) limits their potential for use with irregular or hard-to-reach bone defects. In this context, a ready- and easy-to-use partially demineralized allogeneic BG in a paste form has been developed, with the aim of facilitating such bone grafting procedures. The regenerative properties of this bone paste (BP) was assessed and compared to that of a syngeneic BG in a pre-clinical model of intramembranous bone healing in critical size defects in rat calvaria. The microcomputed tridimensional quantifications and the histological observations at 7 weeks after the implantation revealed that the in vivo bone regeneration of critical-size defects (CSDs) filled with the BP was similar to syngeneic bone grafts (BGs). Thus, this ready-to-use, injectable, and moldable partially demineralized allogeneic BP, displaying equivalent bone healing capacity than the “gold standard,” may be of particular clinical relevance in the context of oral and maxillofacial bone reconstructions.

Articular cartilage (AC) may be affected by many injuries including traumatic lesions that predispose to osteoarthritis. Currently there is no efficient cure for cartilage lesions. In that respect, new strategies for regenerating AC are contemplated with interest. In this context, we aim to develop and characterize an injectable, self-hardening, mechanically reinforced hydrogel (Si-HPCH) composed of silanised hydroxypropymethyl cellulose (Si-HPMC) mixed with silanised chitosan. The in vitro cytocompatibility of Si-HPCH was tested using human adipose stromal cells (hASC). In vivo, we first mixed Si-HPCH with hASC to observe cell viability after implantation in nude mice subcutis. Si-HPCH associated or not with canine ASC (cASC), was then tested for the repair of osteochondral defects in canine femoral condyles. Our data demonstrated that Si-HPCH supports hASC viability in culture. Moreover, Si-HPCH allows the transplantation of hASC in the subcutis of nude mice while maintaining their viability and secretory activity. In the canine osteochondral defect model, while the empty defects were only partially filled with a fibrous tissue, defects filled with Si-HPCH with or without cASC, revealed a significant osteochondral regeneration. To conclude, Si-HPCH is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC as well as the regeneration of osteochondral defects in dogs when implanted alone or with ASC.

Objective: The canonical role of the bone-derived fibroblast growth factor 23 (Fgf23) is to regulate the serum inorganic phosphate (Pi) level. As part of a feedback loop, serum Pi levels control Fgf23 secretion through undefined mechanisms. We recently showed in vitro that the two high-affinity Na+-Pi co-transporters PiT1/Slc20a1 and PiT2/Slc20a2 were required for mediating Pi-dependent signaling. Here, we addressed the contribution of PiT1 and PiT2 to the regulation of Fgf23 secretion. Methods: To this aim, we used PiT2 KO and DMP1Cre; PiT1lox/lox fed Pi-modified diets, as well as ex vivo isolated long bone shafts. Fgf23 secretion and expression of Pi homeostasis-related genes were assessed. Results: In vivo, PiT2 KO mice responded inappropriately to low-Pi diets, displaying abnormally normal serum levels of intact Fgf23. Despite the high iFgf23 level, serum Pi levels remained unaffected, an effect that may relate to lower αKlotho expression in the kidney. Moreover, consistent with a role of PiT2 as a possible endocrine Pi sensor, the iFGF23/cFGF23 ratios were suppressed in PiT2 KO mice, irrespective of the Pi loads. While deletion of PiT1 in osteocytes using the DMP1-Cre mice was inefficient, adenovirus-mediated deletion of PiT1 in isolated long bone shafts suggested that PiT1 does not contribute to Pi-dependent regulation of Fgf23 secretion. In contrast, using isolated bone shafts from PiT2 KO mice, we showed that PiT2 was necessary for the appropriate Pi-dependent secretion of Fgf23, independently from possible endocrine regulatory loops. Conclusions: Our data provide initial mechanistic insights underlying the Pi-dependent regulation of Fgf23 secretion in identifying PiT2 as a potential player in this process, at least in high Pi conditions. Targeting PiT2, therefore, could improve excess FGF23 in hyperphosphatemic conditions such as chronic kidney disease. Keywords: FGF23 secretion, Phosphate sensing, PiT1/Slc20a1, PiT2/Slc20a2, Bone

Discogenic low back pain is considered a major health concern and no etiological treatments are today available to tackle this disease. To clinically address this issue at early stages, there is a rising interest in the stimulation of local cells by in situ injection of growth factors targeting intervertebral disc (IVD) degenerative process. Despite encouraging safety and tolerability results in clinic, growth factors efficacy may be further improved. To this end, the use of a delivery system allowing a sustained release, while protecting growth factors from degradation appears of particular interest. We propose herein the design of a new injectable biphasic system, based on the association of pullulan microbeads (PMBs) into a cellulose-based hydrogel (Si-HPMC), for the TGF-β1 and GDF-5 growth factors sustained delivery. We present for the first time the design and mechanical characterization of both the PMBs and the called biphasic system (PMBs/Si-HPMC). Their loading and release capacities were also studied and we were able to demonstrate a sustained release of both growth factors, for up to 28 days. Noteworthy, the growth factors biological activity on human cells was maintained. Altogether, these data suggest that this PMBs/Si-HPMC biphasic system may be a promising candidate for the development of an innovative bioactive delivery system for IVD regenerative medicine.

The extent of ageing in the musculoskeletal system during the life course affects the quality and length of life. Loss of bone, degraded articular cartilage, and degenerate, narrowed intervertebral discs are primary features of an ageing skeleton, and together they contribute to pain and loss of mobility. This review covers the cellular constituents that make up some key components of the musculoskeletal system and summarizes discussion from the 2015 Aarhus Regenerative Orthopaedic Symposium (AROS) (Regeneration in the Ageing Population) about how each particular cell type alters within the ageing skeletal microenvironment.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

[This corrects the article DOI: 10.1371/journal.pone.0178060.].

Background/Aims: Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. However, chondrogenic differentiation of MSC can ultimately lead to the formation of hypertrophic chondrocytes responsible for the calcification of cartilage. To prevent the production of this calcified matrix at the articular site, the late hypertrophic differentiation of MSCs must be carefully controlled. Given that articular cartilage is avascular, we hypothesized that in addition to its stimulatory role in the early differentiation of chondrogenic cells, hypoxia may prevent their late hypertrophic conversion. Methods: Early and late chondrogenic differentiation were evaluated using human adipose MSC and murine ATDC5 cells cultured under either normoxic (21%O2) or hypoxic (5%O2) conditions. To investigate the effect of hypoxia on late chondrogenic differentiation, the transcriptional activity of hypoxia-inducible factor-1alpha (HIF-1α) and HIF-2α were evaluated using the NoShift DNA-binding assay and through modulation of their activity (chemical inhibitor, RNA interference). Results: Our data demonstrate that low oxygen tension not only stimulates the early chondrogenic commitment of two complementary models of chondrogenic cells, but also inhibits their hypertrophic differentiation. Conclusion: These results suggest that hypoxia can be used as an instrumental tool to prevent the formation of a calcified matrix in MSC-based cartilage tissue engineering.

Mandibular osteoradionecrosis is a severe side effect of radiotherapy after the treatment of squamous cell carcinomas of the upper aerodigestive tract. As an alternative to its treatment by micro-anastomosed free-flaps, preclinical tissular engineering studies have been developed. Total bone marrow (TBM) associated with biphasic calcium phosphate (BCP) significantly enhanced bone formation in irradiated bone. One mechanism, explaining how bone marrow cells can help regenerate tissues like this, is the paracrine effect. The bone marrow cell extract (BMCE) makes use of this paracrine mechanism by keeping only the soluble factors such as growth factors and cytokines. It has provided significant results in repairing various tissues, but has not yet been studied in irradiated bone reconstruction. The purpose of this study was to evaluate the effect of BMCE via an intraosseous or intravenous delivery, with a calcium phosphate scaffold, in irradiated bone reconstruction. Twenty rats were irradiated on their hind limbs with a single 80-Gy dose. Three weeks later, surgery was performed to create osseous defects. The intraosseous group (n = 12) studied the effect of BMCE in situ, with six combinations (empty defect, BCP, TBM, BCP-TBM, lysate only, BCP-lysate). After four different combinations of implantation (empty defect, BCP, TBM, BCP-TBM), the intravenous group (n = 8) received four intravenous injections of BMCE for 2 weeks. Five weeks after implantation, samples were explanted for histological and scanning electron microscopy analysis. Lysate immunogenicity was studied with various mixed lymphocyte reactions. Intravenous injections of BMCE led to a significant new bone formation compared to the intraosseous group. The BCP-TBM mixture remained the most effective in the intraosseous group. However, intravenous injections were more effective, with TBM placed in the defect, with or without biomaterials. Histologically, highly cellularized bone marrow was observed in the defects after intravenous injections, and not after an in situ use of the lysate. The mixed lymphocyte reactions did not show any proliferation after 3, 5, or 7 days of lysate incubation with lymphocytes from another species. This study evaluated the role of BMCE in irradiated bone reconstruction. There were significant results arguing in favor of BMCE intravenous injections. This could open new perspectives to irradiated bone reconstruction.

While therapeutically interesting, the injection of MSCs suffers major limitations including cell death upon injection and a massive leakage outside the injection site. We proposed to entrap MSCs within spherical particles derived from alginate, as a control, or from silanized hydroxypropyl methylcellulose (Si-HPMC). We developed water in an oil dispersion method to produce small Si-HPMC particles with an average size of about 68 μm. We evidenced a faster diffusion of fluorescein isothiocyanate-dextran in Si-HPMC particles than in alginate ones. Human adipose-derived MSCs (hASC) were encapsulated either in alginate or in Si-HPMC, and the cellularized particles were cultured for up to 1 month. Both alginate and Si-HPMC particles supported cell survival, and the average number of encapsulated hASC per alginate and Si-HPMC particle (7102 and 5100, resp.) did not significantly change. The stimulation of encapsulated hASC with proinflammatory cytokines resulted in the production of IDO, PGE2, and HGF whose concentration was always higher when cells were encapsulated in Si-HPMC particles than in alginate ones. We have demonstrated that Si-HPMC and alginate particles support hASC viability and the maintenance of their ability to secrete therapeutic factors.

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-β1 (TGF-β1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-β1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-β1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-β1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.

Chronic Periodontitis (CP) is an inflammatory disease of bacterial origin that results in alveolar bone destruction. Porphyromonas gingivalis (Pg), one of the main periopathogens, initiates an inflammatory cascade by host immune cells thereby increasing recruitment and activity of osteoclasts, the bone resorbing cells, through enhanced production of the crucial osteoclastogenic factor, RANK-L. Antibodies directed against some cytokines (IL-1β, IL-6 and TNF-α) failed to exhibit convincing therapeutic effect in CP. It has been suggested that IL-33, could be of interest in CP.the present study aims to analyze whether and how IL-33 and RANK-L and/or their interplay are involved in the bone destruction associated to CP.mRNAs and protein expressions of IL-33 and RANK-L were analyzed in healthy and CP human gingival samples by immunohistochemistry (IHC) and RT-qPCR. Murine experimental periodontitis (EP) was induced using Pg infected ligature and Pg free ligature around the first maxillary molar. Alveolar bone loss was recorded by μCT. Mouse gingival explants were stimulated for 24 hours with IL-33 and RANK-L mRNA expression investigated by RT-qPCR. Human oral epithelial cells were infected by Pg for 6, 12; 24 hours and IL-33 and RANK-L mRNA expressions were analyzed by RT-qPCR.IL-33 is overexpressed in gingival epithelial cells in human affected by CP as in the murine EP. In human as in murine gingival cells, RANK-L was independently induced by Pg and IL-33. We also showed that the Pg-dependent RANK-L expression in gingival epithelial cells occured earlier than that of IL-33.Our results evidence that IL-33 overexpression in gingival epithelial cells is associated with CP and may trigger RANK-L expression in addition to a direct effect of Pg. Finally, IL-33 may act as an extracellular alarmin (danger signal) showing proinflammatory properties in CP perpetuating bone resorption induced by Pg infection.

The formation of hydroxyapatite crystals and their insertion into collagen fibrils of the matrix are essential steps for bone mineralization. As phosphate is a main structural component of apatite crystals, its uptake by skeletal cells is critical and must be controlled by specialized membrane proteins. In mammals, in vitro studies have suggested that the high-affinity sodium-phosphate cotransporter PiT1 could play this role. In vivo, PiT1 expression was detected in hypertrophic chondrocytes of murine metatarsals, but its implication in bone physiology is not yet deciphered. As the complete deletion of PiT1 results in embryonic lethality at E12.5, we took advantage of a mouse model bearing two copies of PiT1 hypomorphic alleles to study the effect of a low expression of PiT1 on bone mineralization in vivo. In this report, we show that a 85% down-regulation of PiT1 in long bones resulted in a slight (6%) but significant reduction of femur length in young mice (15- and 30-day-old). However, despite a defect in alcian blue / alizarin red S and Von Kossa staining of hypomorphic 1-day-old mice, using X-rays micro-computed tomography, energy dispersive X-ray spectroscopy and histological staining techniques we could not detect differences between hypomorphic and wild-type mice of 15- to 300-days old. Interestingly, the expression of PiT2, the paralog of PiT1, was increased 2-fold in bone of PiT1 hypomorphic mice accounting for a normal phosphate uptake in mutant cells. Whether this may contribute to the absence of bone mineralization defects remains to be further deciphered.

PURPOSE: Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair. METHODS: MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O2. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O2 was finally evaluated using real-time PCR. RESULTS/CONCLUSIONS: 5% O2 increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O2. These data together indicate that although 5% O2 enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.

PURPOSE: Autologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an "all-in-one" study. METHODS: We used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy. RESULTS/CONCLUSIONS: Bone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

Osteoporosis is a bone pathology leading to increase fractures risk and challenging quality of life. Since current treatments could exhibit deleterious side effects, the use of food compounds derived from plants represents a promising innovative alternative due to their potential therapeutic and preventive activities against human diseases. In this study, we investigated the ability of the polyphenol fisetin to counter osteoporosis and analyzed the cellular and molecular mechanisms involved. In vivo, fisetin consumption significantly prevented bone loss in estrogen deficiency and inflammation mice osteoporosis models. Indeed, bone mineral density, micro-architecture parameters and bone markers were positively modulated by fisetin. Consistent with in vivo results, we showed that fisetin represses RANKL-induced osteoclast differentiation and activity as demonstrated by an inhibition of multinucleated cells formation, TRAP activity and differentiation genes expression. The signaling pathways NF-κB, p38 MAPK, JNK and the key transcription factors c-Fos and NFATc1 expressions induced by RANKL, were negatively regulated by fisetin. We further showed that fisetin inhibits the constitutive proteasomal degradation of MKP-1, the phosphatase that deactivates p38 and JNK. Consistently, using shRNA stable cell lines, we demonstrated that impairment of MKP-1 decreases fisetin potency. Taken together, these results strongly support that fisetin should be further considered as a bone protective agent.

BACKGROUND: To improve the efficacy of bone marrow-derived mesenchymal stem cell (MSC) therapy targeted to infarcted myocardium, we investigated whether a self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel seeded with MSC (MSC+hydrogel) could preserve cardiac function and attenuate left ventricular (LV) remodeling during an 8-week follow-up study in a rat model of myocardial infarction (MI). METHODOLOGY/PRINCIPAL FINDING: Si-HPMC hydrogel alone, MSC alone or MSC+hydrogel were injected into the myocardium immediately after coronary artery ligation in female Lewis rats. Animals in the MSC+hydrogel group showed an increase in cardiac function up to 28 days after MI and a mid-term prevention of cardiac function alteration at day 56. Histological analyses indicated that the injection of MSC+hydrogel induced a decrease in MI size and an increase in scar thickness and ultimately limited the transmural extent of MI. These findings show that intramyocardial injection of MSC+hydrogel induced short-term recovery of ventricular function and mid-term attenuation of remodeling after MI. CONCLUSION/SIGNIFICANCE: These beneficial effects may be related to the specific scaffolding properties of the Si-HPMC hydrogel that may provide the ability to support MSC injection and engraftment within myocardium.

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.