Mechanistic Illustration: How Newly-Formed Blood Vessels Stopped by the Mineral Blocks of Bone Substitutes Can Be Avoided by Using Innovative Combined Therapeutics.

One major limitation for the vascularization of bone substitutes used for filling is the presence of mineral blocks. The newly-formed blood vessels are stopped or have to circumvent the mineral blocks, resulting in inefficient delivery of oxygen and nutrients to the implant. This leads to necrosis within the implant and to poor engraftment of the bone substitute. The aim of the present study is to provide a bone substitute currently used in the clinic with suitably guided vascularization properties. This therapeutic hybrid bone filling, containing a mineral and a polymeric component, is fortified with pro-angiogenic smart nano-therapeutics that allow the release of angiogenic molecules. Our data showed that the improved vasculature within the implant promoted new bone formation and that the newly-formed bone swapped the mineral blocks of the bone substitutes much more efficiently than in non-functionalized bone substitutes. Therefore, we demonstrated that our therapeutic bone substitute is an advanced therapeutical medicinal product, with great potential to recuperate and guide vascularization that is stopped by mineral blocks, and can improve the regeneration of critical-sized bone defects. We have also elucidated the mechanism to understand how the newly-formed vessels can no longer encounter mineral blocks and pursue their course of vasculature, giving our advanced therapeutical bone filling great potential to be used in many applications, by combining filling and nano-regenerative medicine that currently fall short because of problems related to the lack of oxygen and nutrients.

Osteochondral repair combining therapeutics implant with mesenchymal stem cells spheroids.

Functional articular cartilage regeneration remains challenging, and it is essential to restore focal osteochondral defects and prevent secondary osteoarthritis. Combining autologous stem cells with therapeutic medical device, we developed a bi-compartmented implant that could promote both articular cartilage and subchondral bone regeneration. The first compartment based on therapeutic collagen associated with bone morphogenetic protein 2, provides structural support and promotes subchondral bone regeneration. The second compartment contains bone marrow-derived mesenchymal stem cell spheroids to support the regeneration of the articular cartilage. Six-month post-implantation, the regenerated articular cartilage surface was 3 times larger than that of untreated animals, and the regeneration of the osteochondral tissue occurred during the formation of hyaline-like cartilage. Our results demonstrate the positive impact of this combined advanced therapy medicinal product, meeting the needs of promising osteochondral regeneration in critical size articular defects in a large animal model combining not only therapeutic implant but also stem cells.

Preclinical safety study of a combined therapeutic bone wound dressing for osteoarticular regeneration.

The extended life expectancy and the raise of accidental trauma call for an increase of osteoarticular surgical procedures. Arthroplasty, the main clinical option to treat osteoarticular lesions, has limitations and drawbacks. In this manuscript, we test the preclinical safety of the innovative implant ARTiCAR for the treatment of osteoarticular lesions. Thanks to the combination of two advanced therapy medicinal products, a polymeric nanofibrous bone wound dressing and bone marrow-derived mesenchymal stem cells, the ARTiCAR promotes both subchondral bone and cartilage regeneration. In this work, the ARTiCAR shows 1) the feasibility in treating osteochondral defects in a large animal model, 2) the possibility to monitor non-invasively the healing process and 3) the overall safety in two animal models under GLP preclinical standards. Our data indicate the preclinical safety of ARTiCAR according to the international regulatory guidelines; the ARTiCAR could therefore undergo phase I clinical trial.

Porphyromonas gingivalis bypasses epithelial barrier and modulates fibroblastic inflammatory response in an in vitro 3D spheroid model.

Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin ß1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC-FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.

Bone substitutes: a review of their characteristics, clinical use, and perspectives for large bone defects management.

Bone replacement might have been practiced for centuries with various materials of natural origin, but had rarely met success until the late 19th century. Nowadays, many different bone substitutes can be used. They can be either derived from biological products such as demineralized bone matrix, platelet-rich plasma, hydroxyapatite, adjunction of growth factors (like bone morphogenetic protein) or synthetic such as calcium sulfate, tri-calcium phosphate ceramics, bioactive glasses, or polymer-based substitutes. All these substitutes are not suitable for every clinical use, and they have to be chosen selectively depending on their purpose. Thus, this review aims to highlight the principal characteristics of the most commonly used bone substitutes and to give some directions concerning their clinical use, as spine fusion, open-wedge tibial osteotomy, long bone fracture, oral and maxillofacial surgery, or periodontal treatments. However, the main limitations to bone substitutes use remain the management of large defects and the lack of vascularization in their central part, which is likely to appear following their utilization. In the field of bone tissue engineering, developing porous synthetic substitutes able to support a faster and a wider vascularization within their structure seems to be a promising way of research.

Synthesis of a Novel Electrospun Polycaprolactone Scaffold Functionalized with Ibuprofen for Periodontal Regeneration: An In Vitro andIn Vivo Study.

Ibuprofen (IBU) has been shown to improve periodontal treatment outcomes. The aim of this study was to develop a new anti-inflammatory scaffold by functionalizing an electrospun nanofibrous poly-ε-caprolactone membrane with IBU (IBU-PCL) and to evaluate its impact on periodontal inflammation, wound healing and regeneration in vitro and in vivo. IBU-PCL was synthesized through electrospinning. The effects of IBU-PCL on the proliferation and migration of epithelial cells (EC) and fibroblasts (FB) exposed to Porphyromonas gingivlais lipopolysaccharide (Pg-LPS) were evaluated through the AlamarBlue test and scratch assay, respectively. Anti-inflammatory and remodeling properties were investigated through Real time qPCR. Finally, the in vivo efficacy of the IBU-PCL membrane was assessed in an experimental periodontitis mouse model through histomorphometric analysis. The results showed that the anti-inflammatory effects of IBU on gingival cells were effectively amplified using the functionalized membrane. IBU-PCL reduced the proliferation and migration of cells challenged by Pg-LPS, as well as the expression of fibronectin-1, collagen-IV, integrin α3ß1 and laminin-5. In vivo, the membranes significantly improved the clinical attachment and IBU-PCL also reduced inflammation-induced bone destruction. These data showed that the IBU-PCL membrane could efficiently and differentially control inflammatory and migratory gingival cell responses and potentially promote periodontal regeneration.

Temporomandibular Joint Regenerative Medicine.

The temporomandibular joint (TMJ) is an articulation formed between the temporal bone and the mandibular condyle which is commonly affected. These affections are often so painful during fundamental oral activities that patients have lower quality of life. Limitations of therapeutics for severe TMJ diseases have led to increased interest in regenerative strategies combining stem cells, implantable scaffolds and well-targeting bioactive molecules. To succeed in functional and structural regeneration of TMJ is very challenging. Innovative strategies and biomaterials are absolutely crucial because TMJ can be considered as one of the most difficult tissues to regenerate due to its limited healing capacity, its unique histological and structural properties and the necessity for long-term prevention of its ossified or fibrous adhesions. The ideal approach for TMJ regeneration is a unique scaffold functionalized with an osteochondral molecular gradient containing a single stem cell population able to undergo osteogenic and chondrogenic differentiation such as BMSCs, ADSCs or DPSCs. The key for this complex regeneration is the functionalization with active molecules such as IGF-1, TGF-ß1 or bFGF. This regeneration can be optimized by nano/micro-assisted functionalization and by spatiotemporal drug delivery systems orchestrating the 3D formation of TMJ tissues.

Semaphorin 3A receptor inhibitor as a novel therapeutic to promote innervation of bioengineered teeth.

The sensory innervation of the dental pulp is essential for tooth function and protection. It is mediated by axons originating from the trigeminal ganglia and is spatio-temporally regulated. We have previously shown that the innervation of bioengineered teeth can be achieved only under immunosuppressive conditions. The aim of this study was to develop a model to determine the role of Semaphorin 3A (Sema3A) in the innervation of bioengineered teeth. We first analysed innervation of the dental pulp of mandibular first molars in newborn (postnatal day 0: PN0) mice deficient for Sema3A (Sema3A-/- ), a strong inhibitor of axon growth. While at PN0, axons detected by immunostaining for peripherin and NF200 were restricted to the peridental mesenchyme in Sema3A+/+ mice, they entered the dental pulp in Sema3A-/- mice. Then, we have implanted cultured teeth obtained from embryonic day-14 (E14) molar germs of Sema3A-/- mice together with trigeminal ganglia. The dental pulps of E14 cultured and implanted Sema3A-/- teeth were innervated, whereas the axons did not enter the pulp of E14 Sema3A+/+ cultured and implanted teeth. A "Membrane Targeting Peptide NRP1," suppressing the inhibitory effect of Sema3A, has been previously identified. The injection of this peptide at the site of implantation allowed the innervation of the dental pulp of bioengineered teeth obtained from E14 dental dissociated mesenchymal and epithelial cells reassociations of ICR mice. In conclusion, these data show that inhibition of only one axon repellent molecule, Sema3A, allows for pulp innervation of bioengineered teeth.

Anti-inflammatory effect of active nanofibrous polymeric membrane bearing nanocontainers of atorvastatin complexes.

AIM: We developed polymeric membranes for local administration of nonsoluble anti-inflammatory statin, as potential wound patch in rheumatic joint or periodontal lesions. METHODS: Electrospun polycaprolactone membranes were fitted with polysaccharide-atorvastatin nanoreservoirs by using complexes with poly-aminocyclodextrin. Characterization methods are UV-Visible and X-ray photoelectron spectroscopy, molecular dynamics, scanning and transmission electron microscopy. In vitro, membranes were seeded with macrophages, and inflammatory cytokine expression were monitored. RESULTS & CONCLUSION: Stable inclusion complexes were formed in solution (1:1 stability constant 368 M-1, -117.40 kJ mol-1), with supramolecular globular organization (100 nm, substructure 30 nm). Nanoreservoir technology leads to homogeneous distribution of atorvastatin calcium trihydrate complexes in the membrane. Quantity embedded was estimated (70-90 µg in 30 µm × 6 mm membrane). Anti-inflammatory effect by cell contact-dependent release reached 60% inhibition for TNF-α and 80% for IL-6. The novelty resides in the double protection offered by the cyclodextrins as drug molecular chaperones, with further embedding into biodegradable nanoreservoirs. The strategy is versatile and can target other diseases.

Hybrid collagen sponge and stem cells as a new combined scaffold able to induce the re-organization of endothelial cells into clustered networks.

The time needed to obtain functional regenerated bone tissue depends on the existence of a reliable vascular support. Current techniques used in clinic, for example after tooth extraction, do not allow regaining or preserving the same bone volume. Our aim is to develop a cellularized active implant of the third generation, equipped with human mesenchymal stem cells to improve the quality of implant vascularization. We seeded a commercialized collagen implant with human mesenchymal stem cells (hMSCs) and then with human umbilical vein endothelial cells (HUVECs). We analyzed the biocompatibility and the behavior of endothelial cells with this implant. We observed a biocompatibility of the active implant, and a re-organization of endothelial cells into clustered networks. This work shows the possibility to develop an implant of the third generation supporting vascularization, improving the medical care of patients.

Nanoengineered implant as a new platform for regenerative nanomedicine using 3D well-organized human cell spheroids.

In tissue engineering, it is still rare today to see clinically transferable strategies for tissue-engineered graft production that conclusively offer better tissue regeneration than the already existing technologies, decreased recovery times, and less risk of complications. Here a novel tissue-engineering concept is presented for the production of living bone implants combining 1) a nanofibrous and microporous implant as cell colonization matrix and 2) 3D bone cell spheroids. This combination, double 3D implants, shows clinical relevant thicknesses for the treatment of an early stage of bone lesions before the need of bone substitutes. The strategy presented here shows a complete closure of a defect in nude mice calvaria after only 31 days. As a novel strategy for bone regenerative nanomedicine, it holds great promises to enhance the therapeutic efficacy of living bone implants.

Smart Implants as a Novel Strategy to Regenerate Well-Founded Cartilage.

Here we explore a new generation of smart, living implants, combining not only active therapeutics but also stem cells, as a novel strategy to regenerate stabilised cartilage and avoid prostheses. This process can regenerate the subchondral bone foundation, which is currently difficult in the clinic.

Advanced nanostructured medical device combining mesenchymal cells and VEGF nanoparticles for enhanced engineered tissue vascularization.

AIM: Success of functional vascularized tissue repair depends on vascular support system supply and still remains challenging. Our objective was to develop a nanoactive implant enhancing endothelial cell activity, particularly for bone tissue engineering in the regenerative medicine field. MATERIALS & METHODS: We developed a new strategy of tridimensional implant based on cell-dependent sustained release of VEGF nanoparticles. These nanoparticles were homogeneously distributed within nanoreservoirs onto the porous scaffold, with quicker reorganization of endothelial cells. Moreover, the activity of this active smart implant on cells was also modulated by addition of osteoblastic cells. RESULTS & CONCLUSION: This sophisticated active strategy should potentiate efficiency of current therapeutic implants for bone repair, avoiding the need for bone substitutes.

Double compartmented and hybrid implant outfitted with well-organized 3D stem cells for osteochondral regenerative nanomedicine.

AIM: Articular cartilage repair remains challenging, because most clinical failures are due to the lack of subchondral bone regeneration. We report an innovative approach improving cartilage repair by regenerating a robust subchondral bone, supporting articular cartilage. MATERIALS & METHODS: We developed a compartmented living implant containing triple-3D structure: stem cells as microtissues for embryonic endochondral development mimic, nanofibrous collagen to enhance mineralization for subchondral bone and alginate hydrogel for cartilage regeneration. RESULTS & CONCLUSION: This system mimics the natural gradient of the osteochondral unit, using only one kind of stem cell, targeting their ability to express specific bone or cartilage proteins. Mineralization gradient of articular cartilage and the natural 'glue' between subchondral bone and cartilage were reproduced in vitro.

Active implant combining human stem cell microtissues and growth factors for bone-regenerative nanomedicine.

AIMS: Mesenchymal stem cells (MSCs) from adult bone marrow provide an exciting and promising stem cell population for the repair of bone in skeletal diseases. Here, we describe a new generation of collagen nanofiber implant functionalized with growth factor BMP-7 nanoreservoirs and equipped with human MSC microtissues (MTs) for regenerative nanomedicine. MATERIALS & METHODS: By using a 3D nanofibrous collagen membrane and by adding MTs rather than single cells, we optimize the microenvironment for cell colonization, differentiation and growth. RESULTS & CONCLUSION: Furthermore, in this study, we have shown that by combining BMP-7 with these MSC MTs in this double 3D environment, we further accelerate bone growth in vivo. The strategy described here should enhance the efficiency of therapeutic implants compared with current simplistic approaches used in the clinic today based on collagen implants soaked in bone morphogenic proteins.

A living thick nanofibrous implant bifunctionalized with active growth factor and stem cells for bone regeneration.

New-generation implants focus on robust, durable, and rapid tissue regeneration to shorten recovery times and decrease risks of postoperative complications for patients. Herein, we describe a new-generation thick nanofibrous implant functionalized with active containers of growth factors and stem cells for regenerative nanomedicine. A thick electrospun poly(ε-caprolactone) nanofibrous implant (from 700 µm to 1 cm thick) was functionalized with chitosan and bone morphogenetic protein BMP-7 as growth factor using layer-by-layer technology, producing fish scale-like chitosan/BMP-7 nanoreservoirs. This extracellular matrix-mimicking scaffold enabled in vitro colonization and bone regeneration by human primary osteoblasts, as shown by expression of osteocalcin, osteopontin, and bone sialoprotein (BSPII), 21 days after seeding. In vivo implantation in mouse calvaria defects showed significantly more newly mineralized extracellular matrix in the functionalized implant compared to a bare scaffold after 30 days' implantation, as shown by histological scanning electron microscopy/energy dispersive X-ray microscopy study and calcein injection. We have as well bifunctionalized our BMP-7 therapeutic implant by adding human mesenchymal stem cells (hMSCs). The activity of this BMP-7-functionalized implant was again further enhanced by the addition of hMSCs to the implant (living materials), in vivo, as demonstrated by the analysis of new bone formation and calcification after 30 days' implantation in mice with calvaria defects. Therefore, implants functionalized with BMP-7 nanocontainers associated with hMSCs can act as an accelerator of in vivo bone mineralization and regeneration.

Integrating Microtissues in Nanofiber Scaffolds for Regenerative Nanomedicine.

A new generation of biomaterials focus on smart materials incorporating cells. Here, we describe a novel generation of synthetic nanofibrous implant functionalized with living microtissues for regenerative nanomedicine. The strategy designed here enhances the effectiveness of therapeutic implants compared to current approaches used in the clinic today based on single cells added to the implant.

Active Nanofibrous Membrane Effects on Gingival Cell Inflammatory Response.

Alpha-melanocyte stimulating hormone (α-MSH) is involved in normal skin wound healing and also has anti-inflammatory properties. The association of α-MSH to polyelectrolyte layers with various supports has been shown to improve these anti-inflammatory properties. This study aimed to evaluate the effects of nanofibrous membrane functionalized with α-MSH linked to polyelectrolyte layers on gingival cell inflammatory response. Human oral epithelial cells (EC) and fibroblasts (FB) were cultured on plastic or electrospun Poly-#-caprolactone (PCL) membranes with α-MSH covalently coupled to Poly-L-glutamic acid (PGA-α-MSH), for 6 to 24 h. Cells were incubated with or without Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Cell proliferation and migration were determined using AlamarBlue test and scratch assay. Expression of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and transforming growth factor-beta (TGF-ß) was evaluated using RT-qPCR method. Cell cultures on plastic showed that PGA-α-MSH reduced EC and FB migration and decreased IL-6 and TGF-ß expression in Pg-LPS stimulated EC. PGA-α-MSH functionalized PCL membranes reduced proliferation of Pg-LPS stimulated EC and FB. A significant decrease of IL-6, TNF-α, and TGF-ß expression was also observed in Pg-LPS stimulated EC and FB. This study showed that the functionalization of nanofibrous PCL membranes efficiently amplified the anti-inflammatory effect of PGA-α-MSH on gingival cells.

Active Nanomaterials to Meet the Challenge of Dental Pulp Regeneration.

The vitality of the pulp is fundamental to the functional life of the tooth. For this aim, active and living biomaterials are required to avoid the current drastic treatment, which is the removal of all the cellular and molecular content regardless of its regenerative potential. The regeneration of the pulp tissue is the dream of many generations of dental surgeons and will revolutionize clinical practices. Recently, the potential of the regenerative medicine field suggests that it would be possible to achieve such complex regeneration. Indeed, three crucial steps are needed: the control of infection and inflammation and the regeneration of lost pulp tissues. For regenerative medicine, in particular for dental pulp regeneration, the use of nano-structured biomaterials becomes decisive. Nano-designed materials allow the concentration of many different functions in a small volume, the increase in the quality of targeting, as well as the control of cost and delivery of active molecules. Nanomaterials based on extracellular mimetic nanostructure and functionalized with multi-active therapeutics appear essential to reverse infection and inflammation and concomitantly to orchestrate pulp cell colonization and differentiation. This novel generation of nanomaterials seems very promising to meet the challenge of the complex dental pulp regeneration.

Nanostructured thick 3D nanofibrous scaffold can induce bone.

Designing unique nanostructured biomimetic materials is a new challenge in modern regenerative medicine. In order to develop functional substitutes for damaged organs or tissues, several methods have been used to create implants able to regenerate robust and durable bone. Electrospinning produces nonwoven scaffolds based on polymer nanofibers mimicking the fibrillar organization of bone extracellular matrix. Here, we describe a biomimetic 3D thick nanofibrous scaffold obtained by electrospinning of the biodegradable, bioresorbable and FDA-approved polymer, poly(ε-caprolactone). Such scaffold presents a thickness reaching one centimeter. We report here the demonstration that the designed nanostructured implant is able to induce in vivo bone regeneration.