Enriching a cellulose hydrogel with a biologically active marine exopolysaccharide for cell-based cartilage engineering.

The development of biologically and mechanically competent hydrogels is a prerequisite in cartilage engineering. We recently demonstrated that a marine exopolysaccharide, GY785, stimulates the in vitro chondrogenesis of adipose stromal cells. In the present study, we thus hypothesized that enriching our silated hydroxypropyl methylcellulose hydrogel (Si-HPMC) with GY785 might offer new prospects in the development of scaffolds for cartilage regeneration. The interaction properties of GY785 with growth factors was tested by surface plasmon resonance (SPR). The biocompatibility of Si-HPMC/GY785 towards rabbit articular chondrocytes (RACs) and its ability to maintain and recover a chondrocytic phenotype were then evaluated in vitro by MTS assay, cell counting and qRT-PCR. Finally, we evaluated the potential of Si-HPMC/GY785 associated with RACs to form cartilaginous tissue in vivo by transplantation into the subcutis of nude mice for 3 weeks. Our SPR data indicated that GY785 was able to physically interact with BMP-2 and TGFß. Our analyses also showed that three-dimensionally (3D)-cultured RACs into Si-HPMC/GY785 strongly expressed type II collagen (COL2) and aggrecan transcripts when compared to Si-HPMC alone. In addition, RACs also produced large amounts of extracellular matrix (ECM) containing glycosaminoglycans (GAG) and COL2. When dedifferentiated RACs were replaced in 3D in Si-HPMC/GY785, the expressions of COL2 and aggrecan transcripts were recovered and that of type I collagen decreased. Immunohistological analyses of Si-HPMC/GY785 constructs transplanted into nude mice revealed the production of a cartilage-like extracellular matrix (ECM) containing high amounts of GAG and COL2. These results indicate that GY785-enriched Si-HPMC appears to be a promising hydrogel for cartilage tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

BMP-2 delivered from a self-crosslinkable CaP/hydrogel construct promotes bone regeneration in a critical-size segmental defect model of non-union in dogs.

OBJECTIVES: To determine whether the addition of recombinant human bone morphogenetic protein (rhBMP-2) to a self-crosslinkable cellulosic hydrogel/biphasic calcium phosphate (BCP) granules construct promotes bone healing in critical-size ulnar defects in dogs. METHODS: A standardized 2 cm long ulnar ostectomy was performed bilaterally in five dogs to compare bone healing with hydrogel/BCP constructs associated with or without rhBMP-2. Cancellous-bone autografts were used as positive controls in unilateral ulnar defects in five additional dogs. Radiographically, bone healing was evaluated at four, eight, 12, 16 and 20 weeks postoperatively. Histological qualitative analysis with microCT imaging and light and scanning electron microscopy were performed 20 weeks after implantation. RESULTS: All rhBMP-2-loaded constructs induced the formation of well-differentiated mineralized lamellar bone surrounding the BCP granules and bridging bone/implant interfaces as early as eight weeks after surgery. Bone regeneration appeared to develop earlier with the rhBMP-2 constructs than with the cancellous-bone autografts while similar results were obtained at 20 weeks. Constructs without any rhBMP-2 showed osteoconductive properties limited to the bone junctions and a lack of osteoinduction without bone ingrowth within the implantation site. In one dog, the leakage of the hydrogel loaded with rhBMP-2 induced an extensive heterotopic bone formation. CLINICAL SIGNIFICANCE: The addition of rhBMP-2 to a self-crosslinkable hydrogel/BCP construct could promote bone regeneration in a critical-size-defect model with similar performance to autologous bone grafts.

Cartilage tissue engineering: From hydrogel to mesenchymal stem cells.

Articular cartilage does not repair itself spontaneously. To promote its repair, the transfer of stem cells from adipose tissue (ATSC) using an injectable self-setting cellulosic-hydrogel (Si-HPMC) appears promising. In this context, the objective of this work was to investigate the influence of in vitro chondrogenic differentiation of ATSC on the in vivo cartilage formation when combined with Si-HPMC. In a first set of experiments, we characterized ATSC for their ability to proliferate, self renew and express typical mesenchymal stem cell surface markers. Then, the potential of ATSC to differentiate towards the chondrogenic lineage and the optimal culture conditions to drive this differentiation were evaluated. Real-time RT-PCR and histological analysis for sulphated glycosaminoglycans and type II collagen revealed that 3-dimensional culture and hypoxic condition favored ATSC chondrogenesis regarding mRNA expression level and the corresponding proteins production. In order to assess the phenotypic stability of chondrogenically-differentiated ATSC, real-time RT-PCR for specific terminal chondrogenic markers and alkaline phosphatase activity assay were performed. In addition to promote chondrogenesis, our culture conditions seem to prevent the terminal differentiation of ATSC. Histological examination of ATSC/Si-HPMC implants suggested that the in vitro chondrogenic pre-commitment of ATSC in monolayer is sufficient to obtain cartilaginous tissue in vivo.

Effects of autologous platelet rich plasma gel and calcium phosphate biomaterials on bone healing in an ulnar ostectomy model in dogs.

The aim of the study was to evaluate the bone healing properties of an osteopromotive platelet rich plasma (PRP) gel in combination with osteoconductive calcium phosphate (CaP) ceramic granules in a long-bone critical size defect in dogs. A standardised 2 cm long ulnar ostectomy was performed bilaterally in four dogs to compare new-bone formation by CaP matrix with and without association with PRP. Radiographic and histological evaluations were performed blindly. Radiographic evaluation was performed at three, six, nine, 12 and 16 weeks postoperatively. Quantitative measurements of new-bone formation were compared using statistical analysis. At explantation 16 weeks after surgery, no significant ossification was present, neither with CaP granules alone nor in association with PRP gel, and there was no difference of radiodensity between the groups. Qualitative histological evaluation demonstrated for both types of implants the presence of non-mineralised fibrous connective tissue around the CaP granules. New-bone formation was only present to a very small extent within the macropores of the CaP granules at the distal bone-implant interface. In our model which exhibited very limited osteoconduction, neither the CaP granules alone nor in association with PRP were sufficient to stimulate bone healing. In this canine model employing a critical size ulnar gap, the combination of CaP granules and PRP did not effectively promote bone regeneration.

An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects.

Articular cartilage has a low capacity for spontaneous repair. To promote the repair of this tissue, the transfer of autologous chondrocytes using a three-dimensional matrix appears promising. In this context, the aim of the present work was to investigate the potential use of autologous rabbit nasal chondrocytes (RNC) associated with an injectable self-setting cellulose-based hydrogel (Si-HPMC). Firstly, the influence of Si-HPMC on chondrocytic phenotype was investigated by real-time PCR for specific chondrocyte markers (type II collagen and aggrecan) and type I collagen. Thereafter, autologous RNC were amplified in vitro for 4 weeks before transplantation with Si-HPMC into a rabbit articular cartilage defect followed by analysis 6 weeks later. Implants were histologically characterized for the presence of sulfated GAG and type II collagen. Transcripts analysis indicated that dedifferentiated RNC recovered expression of the main chondrocytic markers after in vitro three-dimensional culture within Si-HPMC. Histological analysis of autologous RNC transplanted in an articular cartilage defect revealed the formation of repair tissue with a histological organization similar to that of healthy articular cartilage. In addition, immunohistological analysis of type II collagen suggested that the repair tissue was a hyaline-like cartilage. Si-HPMC hydrogel associated with nasal chondrocytes therefore appears a promising injectable tissue engineering device for the repair of articular cartilage.

Nasal chondrocytes and fibrin sealant for cartilage tissue engineering.

Hybrid constructs associating a biodegradable matrix and autologous chondrocytes hold promise for the treatment of articular cartilage defects. In this context, our objective was to investigate the potential use of nasal chondrocytes associated with a fibrin sealant for the treatment of articular cartilage defects. The phenotype of primary nasal chondrocytes (NC) from human (HNC) and rabbit (RNC) origin were characterized by RT-PCR. The ability of constructs associating fibrin sealant and NC to form a cartilaginous tissue in vivo was investigated, firstly in a subcutaneous site in nude mice and secondly in an articular cartilage defect in rabbit. HNC express type II collagen and aggrecan, the two major hallmarks of a chondrocytic phenotype. Furthermore, when injected subcutaneously into nude mice within a fibrin sealant, these chondrocytes were able to form a cartilage-like tissue. Our data indicate that RNC also express type II collagen and aggrecan and maintained their phenotype in three-dimensional culture within a fibrin sealant. Moreover, treatment of rabbit articular cartilage defects with autologous RNC embedded in a fibrin sealant led to the formation of a hyalin-like repair tissue. The use of fibrin sealant containing hybrid autologous NC therefore appears as a promising approach for cell-based therapy of articular cartilage.