Collateral effects of targeting the nucleus pulposus via a transpedicular or transannular surgical route: a combined X-ray, MRI, and histological long-term descriptive study in sheep.

PURPOSE: In the context of regenerative medicine strategies, based in particular on the injection of regenerative cells, biological factors, or biomaterials into the nucleus pulposus (NP), two main routes are used: the transpedicular approach (TPA) and the transannular approach (TAA). The purpose of our study was to compare the long-term consequences of the TPA and the TAA on intervertebral disc (IVD) health through a longitudinal follow-up in an ovine model. METHODS: The TPA and the TAA were performed on 12 IVDs from 3 sheep. Six discs were left untreated and used as controls. The route and injection feasibility, as well as the IVD environment integrity, were assessed by MRI (T2-weighted signal intensity), micro-CT scan, and histological analyses (Boos' scoring). The sheep were assessed at 1, 3, and 7 months. RESULTS: Both the TPA and the TAA allowed access to the NP. They both induced NP degeneration, as evidenced by a decrease in the T2wsi and an increase in the Boos' scores. The TPA led to persistent end-plate defects and herniation of NP tissue (Schmorl's node-like) after 7 months as well as the presence of osseous fragments in the NP. CONCLUSIONS: The TPA induced more severe lesions in IVDs and vertebrae compared to the TAA. The lesions induced by the TPA are reason to consider whether or not this route is optimal for studying IVD regenerative medicine approaches.

Preliminary evaluation of an osteochondral autograft, a prosthetic implant, and a biphasic absorbable implant for osteochondral reconstruction in a sheep model.

OBJECTIVE: To determine the ability of three implants to enhance the healing of osteochondral defects: (1) a biphasic construct composed of calcium phosphate (CaP) and chitosan/cellulosic polymer, (2) a titanium-polyurethane implant, and (3) an osteochondral autograft. STUDY DESIGN: Experimental study. ANIMALS: Ten adult female sheep. METHODS: In five sheep, an 8-mm diameter osteochondral defect was created on the medial femoral condyle of a stifle and filled with a synthetic titanium-polyurethane implant. In five sheep, a similar defect was filled with an osteochondral autograft, and the donor site was filled with a biphasic construct combining CaP granules and a chitosan/cellulosic polymer. Sheep were monitored daily for lameness. Stifle radiographs and MRI were evaluated at 20 weeks, prior to animals being humanely killed. Surgical sites were evaluated with histology, microcomputed tomography, and scanning electron microscopy. RESULTS: Clinical outcomes were satisfactory regardless of the tested biomaterials. All implants appeared in place on imaging studies. Osteointegration of prosthetic implants varied between sites, with limited ingrowth of new bone into the titanium structure. Autografts and biphasic constructs were consistently well integrated in subchondral bone. All autografts except one contained a cartilage surface, and all biphasic constructs except one partially restored hyaline cartilage surface. CONCLUSION: Biphasic constructs supported hyaline cartilage and subchondral bone regeneration, although restoration of the articular cartilage was incomplete. CLINICAL IMPACT: Biphasic constructs may provide an alternative treatment for osteochondral defects, offering a less invasive approach compared with autologous grafts and eliminating the requirement for a prosthetic implant.

TGF-ß1 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-ß1 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.

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

PURPOSE: To investigate the suitability of the transpedicular approach (TPA) in a sheep model of IVD regenerative strategies METHODS: 24 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. RESULTS: 83% 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. CONCLUSIONS: TPA 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.

Inverse regulation of early and late chondrogenic differentiation by oxygen tension provides cues for stem cell-based cartilage tissue engineering.

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.

Pharmacological modulation of human mesenchymal stem cell chondrogenesis by a chemically oversulfated polysaccharide of marine origin: potential application to cartilage regenerative medicine.

Mesenchymal stem cells (MSCs) are considered as an attractive source of cells for cartilage engineering due to their availability and capacity for expansion and multipotency. Differentiation of MSC into chondrocytes is crucial to successful cartilage regeneration and can be induced by various biological agents, including polysaccharides that participate in many biological processes through interactions with growth factors. Here, we hypothesize that growth factor-induced differentiation of MSC can be increased by chemically oversulfated marine polysaccharides. To test our hypothesis, human adipose tissue-derived MSCs (hATSCs) were cultured in pellets with transforming growth factor (TGF)-ß1-supplemented chondrogenic medium containing either the polysaccharide GY785 DR or its oversulfated isoform GY785 DRS. Chondrogenesis was monitored by the measurement of pellet volume, quantification of DNA, collagens, glycosaminoglycans (GAGs), and immunohistological staining. Our data revealed an increase in pellet volume, total collagens, and GAG production with GY785 DRS and chondrogenic medium. The enhanced chondrogenic differentiation of hATSC was further demonstrated by the increased expression of several chondrogenic markers by real-time reverse transcription-polymerase chain reaction. In addition, surface plasmon resonance analyses revealed that TGF-ß1 bound GY785 DRS with higher affinity compared to GY785 DR. In association with TGF-ß1, GY785 DRS was found to upregulate the phosphorylation of extracellular signal-regulated kinase 1/2, indicating that oversulfated polysaccharide affects the mitogen activated protein kinase signaling activity. These results demonstrate the upregulation of TGF-ß1-dependent stem cell chondrogenesis by a chemically oversulfated marine polysaccharide. This polysaccharide of marine origin is easily producible and therefore could be considered a promising additive to drive efficient and reliable MSC chondrogenesis for cartilage tissue engineering.

Injectable Hydrogel Membrane for Guided Bone Regeneration.

In recent years, multicomponent hydrogels such as interpenetrating polymer networks (IPNs) have emerged as innovative biomaterials due to the synergistic combination of the properties of each network. We hypothesized that an innovative non-animal IPN hydrogel combining self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) with photochemically cross-linkable dextran methacrylate (DexMA) could be a valid alternative to porcine collagen membranes in guided bone regeneration. Calvaria critical-size defects in rabbits were filled with synthetic biphasic calcium phosphate granules in conjunction with Si-HPMC; DexMA; or Si-HPMC/DexMA experimental membranes; and in a control group with a porcine collagen membrane. The synergistic effect obtained by interpenetration of the two polymer networks improved the physicochemical properties, and the gel point under visible light was reached instantaneously. Neutral red staining of murine L929 fibroblasts confirmed the cytocompatibility of the IPN. At 8 weeks, the photo-crosslinked membranes induced a similar degree of mineral deposition in the calvaria defects compared to the positive control, with 30.5 ± 5.2% for the IPN and 34.3 ± 8.2% for the collagen membrane. The barrier effect appeared to be similar in the IPN test group compared with the collagen membrane. In conclusion, this novel, easy-to-handle and apply, photochemically cross-linkable IPN hydrogel is an excellent non-animal alternative to porcine collagen membrane in guided bone regeneration procedures.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

Comparison of MRI T1, T2, and T2* mapping with histology for assessment of intervertebral disc degeneration in an ovine model.

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 < 0.05) except for the most ventral rectangular ROI on T2 maps. Correlations were excellent for all of the T1 ROIs and the T2* NP ROIs. Highly significant differences in T1 values were found between all Pfirrmann grades except between grades I/II and between grades III/IV. Significant differences were identified in the T2 and the T2* values between all grades except between grades I/III. T1, T2, and T2* relaxation times measurements of the NP are an accurate and time-efficient tool to assess lumbar IDD in sheep. Variable-flip-angle T1 mapping may be further considered as a valuable method to investigate IDD and to assess the efficacy of regenerative treatments in longitudinal studies.

Characterization of the age-dependent intervertebral disc changes in rabbit by correlation between MRI, histology and gene expression.

BACKGROUND: The present study was conducted to address whether the intervertebral disc of rabbit could be considered (i) as a valuable model to provide new insights into the tissue and cellular changes of Nucleus pulposus aging and (ii) as an appropriate tool to investigate the efficacy of Nucleus pulposus cell-based biotherapies. METHODS: Lumbar intervertebral disc from rabbits with increasing ages (1, 6 and 30 month-old) were compared by MRI and histological observation using Pfirrmann's grading and Boos' scoring respectively. The expression of transcripts (COL2A1, AGC1, COL1A1, MMP13, BMP2, MGP and p21) in Nucleus pulposus cells were analysed by quantitative real-time PCR. RESULTS: MRI analysis indicated an early age-dependent increase in the Pfirrmann's grading. Histological Boos' scoring was also increased. The analysis of transcript expression levels showed that COL2A1 and AGC1 were down-regulated as a function of age. Conversely, COL1A1, MMP-13, BMP-2, MGP and p21 were significantly up-regulated in the Nucleus pulposus cells of aged rabbit intervertebral disc. CONCLUSIONS: Our study describes the consistency of the rabbit as a model of intervertebral disc changes as a function of age by correlating tissue alteration with cellular modification measured.

Injectable silanized hyaluronic acid hydrogel/biphasic calcium phosphate granule composites with improved handling and biodegradability promote bone regeneration in rabbits.

Biphasic calcium phosphate (BCP) granules are osteoconductive biomaterials used in clinics to favor bone reconstruction. Yet, poor cohesivity, injectability and mechanical properties restrain their use as bone fillers. In this study, we incorporated BCP granules into in situ forming silanized hyaluronic acid (Si-HA) and hydroxypropylmethylcellulose (Si-HPMC) hydrogels. Hydrogel composites were shown to be easily injectable (F < 30 N), with fast hardening properties (<5 min), and similar mechanical properties (E∼ 60 kPa). In vivo, both hydrogels were well tolerated by the host, but showed different biodegradability with Si-HA gels being partially degraded after 21d, while Si-HPMC gels remained stable. Both composites were easily injected into critical size rabbit defects and remained cohesive. After 4 weeks, Si-HPMC/BCP led to poor bone healing due to a lack of degradation. Conversely, Si-HA/BCP composites were fully degraded and beneficially influenced bone regeneration by increasing the space available for bone ingrowth, and by accelerating BCP granules turnover. Our study demonstrates that the degradation rate is key to control bone regeneration and that Si-HA/BCP composites are promising biomaterials to regenerate bone defects.

Posttraumatic Osteoarthritis Damage in Mice: From Histological and Micro-Computed Tomodensitometric Changes to Gait Disturbance.

OBJECTIVES: Osteoarthritis is a painful joint disease responsible for walking impairment. Its quantitative assessment by gait analysis in mice may be a relevant and noninvasive strategy to assess the disease severity. In this study, we aimed to determine the severity of osteoarthritis at the tissular and gait levels in unilateral and bilateral posttraumatic murine osteoarthritis. METHODS: Twenty-four C57BL/6 male mice were randomly assigned to 3 groups (n = 8/group): controls, unilateral surgery, and bilateral surgery. Posttraumatic osteoarthritis was induced unilaterally or bilaterally by destabilization of the medial meniscus. Gait analysis was performed weekly with the CatWalkTM XT system until the 16th week after surgery. After animal sacrifices, histological and micro-computed tomographic assessment was performed. RESULTS: Operated knees showed a significant increase in the histological score compared with controls (P < 0.001). Calcified anterior medial meniscal bone volume was higher on the ipsilateral side after unilateral destabilization of the medial meniscus (P < 0.001) and on both sides after bilateral intervention (P < 0.01). One week after surgery, the mice mean speed decreased significantly in both operated groups (P < 0.001 and P < 0.05). In the unilateral group, a significant increase in the contralateral hind print area appeared from week 4 to week 16. CONCLUSIONS: While bilateral destabilization of the medial meniscus induced no detectable gait modification except 1 week after surgery, unilateral model was responsible for a gait disturbance on the contralateral side. Further studies are needed to better define the place of the CatWalkTM in the evaluation of mouse models of osteoarthritis.

A partially demineralized allogeneic bone graft: in vitro osteogenic potential and preclinical evaluation in two different intramembranous bone healing models.

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.

Silanization of Chitosan and Hydrogel Preparation for Skeletal Tissue Engineering.

Tissue engineering is a multidisciplinary field that relies on the development of customized biomaterial to support cell growth, differentiation and matrix production. Toward that goal, we designed the grafting of silane groups onto the chitosan backbone (Si-chito) for the preparation of in situ setting hydrogels in association with silanized hydroxypropyl methylcellulose (Si-HPMC). Once functionalized, the chitosan was characterized, and the presence of silane groups and its ability to gel were demonstrated by rheology that strongly suggests the presence of silane groups. Throughout physicochemical investigations, the Si-HPMC hydrogels containing Si-chito were found to be stiffer with an injection force unmodified. The presence of chitosan within the hydrogel has demonstrated a higher adhesion of the hydrogel onto the surface of tissues. The results of cell viability assays indicated that there was no cytotoxicity of Si-chito hydrogels in 2D and 3D culture of human SW1353 cells and human adipose stromal cells, respectively. Moreover, Si-chito allows the transplantation of human nasal chondrocytes in the subcutis of nude mice while maintaining their viability and extracellular matrix secretory activity. To conclude, Si-chito mixed with Si-HPMC is an injectable, self-setting and cytocompatible hydrogel able to support the in vitro and in vivo viability and activity of hASC.

In Situ Forming, Silanized Hyaluronic Acid Hydrogels with Fine Control Over Mechanical Properties and In Vivo Degradation for Tissue Engineering Applications.

In situ forming hydrogels that can be injected into tissues in a minimally-invasive fashion are appealing as delivery vehicles for tissue engineering applications. Ideally, these hydrogels should have mechanical properties matching those of the host tissue, and a rate of degradation adapted for neo-tissue formation. Here, the development of in situ forming hyaluronic acid hydrogels based on the pH-triggered condensation of silicon alkoxide precursors into siloxanes is reported. Upon solubilization and pH adjustment, the low-viscosity precursor solutions are easily injectable through fine-gauge needles prior to in situ gelation. Tunable mechanical properties (stiffness from 1 to 40 kPa) and associated tunable degradability (from 4 days to more than 3 weeks in vivo) are obtained by varying the degree of silanization (from 4.3% to 57.7%) and molecular weight (120 and 267 kDa) of the hyaluronic acid component. Following cell encapsulation, high cell viability (> 80%) is obtained for at least 7 days. Finally, the in vivo biocompatibility of silanized hyaluronic acid gels is verified in a subcutaneous mouse model and a relationship between the inflammatory response and the crosslink density is observed. Silanized hyaluronic acid hydrogels constitute a tunable hydrogel platform for material-assisted cell therapies and tissue engineering applications.

Tailored Three-Dimensionally Printed Triply Periodic Calcium Phosphate Implants: A Preclinical Study for Craniofacial Bone Repair.

Finding alternative strategies for the regeneration of craniofacial bone defects (CSD), such as combining a synthetic ephemeral calcium phosphate (CaP) implant and/or active substances and cells, would contribute to solving this reconstructive roadblock. However, CaP's architectural features (i.e., architecture and composition) still need to be tailored, and the use of processed stem cells and synthetic active substances (e.g., recombinant human bone morphogenetic protein 2) drastically limits the clinical application of such approaches. Focusing on solutions that are directly transposable to the clinical setting, biphasic calcium phosphate (BCP) and carbonated hydroxyapatite (CHA) 3D-printed disks with a triply periodic minimal structure (TPMS) were implanted in calvarial critical-sized defects (rat model) with or without addition of total bone marrow (TBM). Bone regeneration within the defect was evaluated, and the outcomes were compared to a standard-care procedure based on BCP granules soaked with TBM (positive control). After 7 weeks, de novo bone formation was significantly greater in the CHA disks + TBM group than in the positive controls (3.33 mm3 and 2.15 mm3, respectively, P=0.04). These encouraging results indicate that both CHA and TPMS architectures are potentially advantageous in the repair of CSDs and that this one-step procedure warrants further clinical investigation.

Controlled release of biological factors for endogenous progenitor cell migration and intervertebral disc extracellular matrix remodelling.

The recent description of resident stem/progenitor cells in degenerated intervertebral discs (IVDs) supports the notion that their regenerative capacities could be harnessed to stimulate endogenous repair of the nucleus pulposus (NP). In this study, we developed a delivery system based on pullulan microbeads (PMBs) for sequential release of the chemokine CCL-5 to recruit these disc stem/progenitor cells to the NP tissue, followed by the release of the growth factors TGF-ß1 and GDF-5 to induce the synthesis of a collagen type II- and aggrecan-rich extracellular matrix (ECM). Bioactivity of released CCL5 on human adipose-derived stem cells (hASCs), selected to mimic disc stem/progenitors, was demonstrated using a Transwell® chemotaxis assay. The regenerative effects of loaded PMBs were investigated in ex vivo spontaneously degenerated ovine IVDs. Fluorescent hASCs were seeded on the top cartilaginous endplates (CEPs); the degenerated NPs were injected with PMBs loaded with CCL5, TGF-ß1, and GDF-5; and the IVDs were then cultured for 3, 7, and 28 days to allow for cell migration and disc regeneration. The PMBs exhibited sustained release of biological factors for 21 days. Ex vivo migration of seeded hASCs from the CEP toward the NP was demonstrated, with the cells migrating a significantly greater distance when loaded PMBs were injected (5.8 ± 1.3 mm vs. 3.5 ± 1.8 mm with no injection of PMBs). In ovine IVDs, the overall NP cellularity, the collagen type II and the aggrecan staining intensities, and the Tie2+ progenitor cell density in the NP were increased at day 28 compared to the control groups. Considered together, PMBs loaded with CCL5/TGF-ß1/GDF-5 constitute an innovative and promising strategy for controlled release of growth factors to promote cell recruitment and extracellular matrix remodelling.

A Self-Setting Hydrogel of Silylated Chitosan and Cellulose for the Repair of Osteochondral Defects: From in vitro Characterization to Preclinical Evaluation in Dogs.

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.

In vitro and in vivo evaluation of an electrospun-aligned microfibrous implant for Annulus fibrosus repair.

Annulus fibrosus (AF) impairment is associated with reherniation, discogenic pain, and disc degeneration after surgical partial discectomy. Due to a limited intrinsic healing capacity, defects in the AF persist over time and it is hence necessary to adopt an appropriate strategy to close and repair the damaged AF. In this study, a cell-free biodegradable scaffold made of polycaprolactone (PCL), electrospun, aligned microfibers exhibited high levels of cell colonization, alignment, and AF-like extracellular matrix deposition when evaluated in an explant culture model. The biomimetic multilayer fibrous scaffold was then assessed in an ovine model of AF impairment. After 4 weeks, no dislocation of the implants was detected, and only one sample out of six showed a partial delamination. Histological and immunohistochemical analyses revealed integration of the implant with the surrounding tissue as well as homogeneously aligned collagen fiber organization within each lamella compared to the disorganized and scarcer fibrous tissue in a randomly organized control fibrous scaffold. In conclusion, this biomimetic electrospun implant exhibited promising properties in terms of AF defect closure, with AF-like neotissue formation that fully integrated with the surrounding ovine tissue.

In situ photochemical crosslinking of hydrogel membrane for Guided Tissue Regeneration.

OBJECTIVE: Periodontitis is an inflammatory disease that destroys the tooth-supporting attachment apparatus. Guided tissue regeneration (GTR) is a technique based on a barrier membrane designed to prevent wound space colonization by gingival cells. This study examined a new formulation composed of two polymers that could be photochemically cross-linked in situ into an interpenetrated polymer network (IPN) forming a hydrogel membrane. METHODS: We synthetized and characterized silanized hydroxypropyl methylcellulose (Si-HPMC) for its cell barrier properties and methacrylated carboxymethyl chitosan (MA-CMCS) for its degradable backbone to use in IPN. Hydrogel membranes were cross-linked using riboflavin photoinitiator and a dentistry visible light lamp. The biomaterial's physicochemical and mechanical properties were determined. Hydrogel membrane degradation was evaluated in lysozyme. Cytocompatibility was estimated by neutral red uptake. The cell barrier property was studied culturing human primary gingival fibroblasts or human gingival explants on membrane and analyzed with confocal microscopy and histological staining. RESULTS: The IPN hydrogel membrane was obtained after 120s of irradiation. The IPN showed a synergistic increase in Young moduli compared with the single networks. The CMCS addition in IPN allows a progressive weight loss compared to each polymer network. Cytocompatibility was confirmed by neutral red assay. Human cell invasion was prevented by hydrogel membranes and histological sections revealed that the biomaterial exhibited a barrier effect in contact with soft gingival tissue. SIGNIFICANCE: We demonstrated the ability of an innovative polymer formulation to form in situ, using a dentist's lamp, an IPN hydrogel membrane, which could be an easy-to-use biomaterial for GTR therapy.