Evaluation of a hydrogel membrane on bone regeneration in furcation periodontal defects in dogs.

The aim of the study was to evaluate bone regeneration using a canine model with surgically created periodontal defects filled for 12 weeks using a stratified biomaterial consisting in a biphasic calcium phosphate (BCP) covered with a crosslinking hydrogel acting as polymer membrane of silated hydroxypropyl methylcellulose (Si-HPMC) as the tested new concept. Bilateral, critical-sized, defects were surgically created at the mandibular premolar teeth of six adult beagle dogs. The defects were randomly allocated and: (i) left empty for spontaneous healing or filled with: (ii) BCP and a collagen membrane; (iii) BCP and hydrogel Si-HPMC membrane. At 12 weeks, the experimental conditions resulted in significantly enhanced bone regeneration in the test BCP/Si-HPMC group. Within the limits of this study, we suggest that the hydrogel Si-HPMC may act as an occlusive barrier to protect bone area from soft connective tissue invasion and then effectively contribute to enhance bone regeneration.

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.

Vertebroplasty using bisphosphonate-loaded calcium phosphate cement in a standardized vertebral body bone defect in an osteoporotic sheep model.

In the context of bone regeneration in an osteoporotic environment, the present study describes the development of an approach based on the use of calcium phosphate (CaP) bone substitutes that can promote new bone formation and locally deliver in situ bisphosphonate (BP) directly at the implantation site. The formulation of a CaP material has been optimized by designing an injectable apatitic cement that (i) hardens in situ despite the presence of BP and (ii) provides immediate mechanical properties adapted to clinical applications in an osteoporotic environment. We developed a large animal model for simulating lumbar vertebroplasty through a two-level lateral corpectomy on L3 and L4 vertebrae presenting a standardized osteopenic bone defect that was filled with cements. Both 2-D and 3-D analysis of microarchitectural parameters demonstrated that implantation of BP-loaded cement in such vertebral defects positively influenced the microarchitecture of the adjacent trabecular bone. This biological effect was dependent on the distance from the implant, emphasizing the in situ effect of the BP and its release from the cement. As a drug device combination, this BP-containing apatitic cement shows good promise as a local approach for the prevention of osteoporotic vertebral fractures through percutaneous vertebroplasty procedures.

Effects of in vitro low oxygen tension preconditioning of adipose stromal cells on their in vivo chondrogenic potential: application in cartilage tissue repair.

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-ß) 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.

Laser-treated Nucleus pulposus as an innovative model of intervertebral disc degeneration.

This study describes an innovative experimentally induced model of intervertebral disc degeneration. This innovative approach is based on the induction of extracellular matrix disorders in the intervertebral disc (IVD) using a diode laser. For this study, 15 one-year-old and five 30-month-old New Zealand White rabbits were used. Two procedures were tested to trigger IVD degeneration: needle aspiration (reference technique) and a laser approach. The IVD degeneration process was assessed 20, 40, 60, 90 and 120 days after surgery by X-ray radiography (IVD height), magnetic resonance imaging (MRI) (T2 intensity of IVD signal) and histological analysis using modified Boos' scoring. Our data indicate that a marked IVD degeneration was found compared with sham-operated animals regardless of the procedure tested. A significant decrease in disc height on X-ray radiographs was first demonstrated. In addition, MRI disc signals were significantly reduced in both groups. Finally, a statistically significant increase in Boos' scoring was found in both laser and aspiration-induced IVD degeneration. Interestingly, IVD degeneration induced by laser treatment was more progressive compared with aspiration. Moreover, the histological results indicated that laser-induced disc degeneration was quite similar to that obtained during the natural aging process as observed in 30-month-old rabbits. Our study describes the consistency of this innovative experimentally-induced animal model of IVD degeneration. The radiological, MRI and histological data confirm its relevance. The histological examination indicates that IVD degeneration induced by laser treatment is comparable to the degenerative process observed during the onset of spontaneous IVD degeneration. This model could be a useful tool to help us validate biomaterial-assisted, cell-based, regenerative medicine strategies for the prevention and treatment of IVD degeneration.

The effect of two- and three-dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel.

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.

Macrophage and osteoblast responses to biphasic calcium phosphate microparticles.

The aim of this work was to investigate in vitro the biological events leading to ectopic bone formation in contact with microporous biphasic calcium phosphate (BCP) ceramics. After implantation, microparticles may arise from their degradation and induce an inflammatory response involving macrophages. The secretion of pro-inflammatory cytokines may affect the differentiation of osteoblasts. Mouse macrophage-like (J774) and osteoblast-like (MC3T3-E1) cells were cultured in the presence of BCP microparticles of different sizes (<20, 40-80, or 80-200 microm). The smallest microparticles decreased the viability of both cell types as measured with LDH and methyl tetrazolium salt assays, and enhanced the secretion of pro-inflammatory cytokines (IL-6 and TNF-alpha) by macrophages after 24 h, as revealed by ELISA. Osteoblastic cells were then cultured for 96 h in the presence of these pro-inflammatory cytokines and their differentiation studied by RT-PCR. MC3T3-E1 cells cultured with TNF-alpha showed a decrease in osterix, PTH receptor (PTHR1), and osteocalcin gene expression. On the contrary, IL-6 enhanced the expression of osterix, Runx2, alkaline phosphatase, and osteocalcin compared with plastic. In conclusion, this study shows that the inflammatory response initiated by BCP microparticles may have both detrimental and beneficial effects on osteogenesis.