Cartilage repair using mesenchymal stem cell (MSC) sheet and MSCs-loaded bilayer PLGA scaffold in a rabbit model.

PURPOSE: The integration of regenerated cartilage with surrounding native cartilage is a major challenge for the success of cartilage tissue-engineering strategies. The purpose of this study is to investigate whether incorporation of the power of mesenchymal stem cell (MSC) sheet to MSCs-loaded bilayer poly-(lactic-co-glycolic acid) (PLGA) scaffolds can improve the integration and repair of cartilage defects in a rabbit model. METHODS: Rabbit bone marrow-derived MSCs were cultured and formed cell sheet. Full-thickness cylindrical osteochondral defects (4 mm in diameter, 3 mm in depth) were created in the patellar groove of 18 New Zealand white rabbits and the osteochondral defects were treated with PLGA scaffold (n = 6), PLGA/MSCs (n = 6) or MSC sheet-encapsulated PLGA/MSCs (n = 6). After 6 and 12 weeks, the integration and tissue response were evaluated histologically. RESULTS: The MSC sheet-encapsulated PLGA/MCSs group showed significantly more amounts of hyaline cartilage and higher histological scores than PLGA/MSCs group and PLGA group (P < 0.05). In addition, the MSC sheet-encapsulated PLGA/MCSs group showed the best integration between the repaired cartilage and surrounding normal cartilage and subchondral bone compared to other two groups. CONCLUSIONS: The novel method of incorporation of MSC sheet to PLGA/MCSs could enhance the ability of cartilage regeneration and integration between repair cartilage and the surrounding cartilage. Transplantation of autologous MSC sheet combined with traditional strategies or cartilage debris might provide therapeutic opportunities for improving cartilage regeneration and integration in humans.

Promotion of osteogenic differentiation of stem cells and increase of bone-bonding ability in vivo using urease-treated titanium coated with calcium phosphate and gelatin.

Because of its excellent biocompatibility and low allergenicity, titanium has been widely used for bone replacement and tissue engineering. To produce a desirable composite with enhanced bone response and mechanical strength, in this study bioactive calcium phosphate (CaP) and gelatin composites were coated onto titanium (Ti) via a novel urease technique. The cellular responses to the CaP/gelatin/Ti (CaP/gel/Ti) and bone bonding ability were evaluated with proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) on CaP/gel/Ti and CaP/Ti in vitro. The results showed that the optical density values, alkaline phosphatase expression and genes expression of MSCs on CaP/gel/Ti were similar to those on CaP/Ti, yet significantly higher than those on pure Ti (p < 0.05). CaP/gel/Ti and CaP/Ti rods (2 mm in diameter, 10 mm in length) were also implanted into femoral shaft of rabbits and pure Ti rods served as control (n = 10). Histological examination, scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) measurements were performed at 4 and 8 weeks after the operation. The histological and SEM observations demonstrated clearly that more new bone formed on the surface of CaP/gel/Ti than in the other two groups at each time point. The CaP/gel/Ti bonded to the surrounding bone directly with no intervening soft tissue layer. An interfacial layer, containing Ti, Ca and P, was found to form at the interface between bone and the implant on all three groups by EDS analysis. However, the content of Ca, P in the surface of CaP/gel/Ti implants was more than in the other two groups at each time point. The CaP/gel/Ti modified by the urease method was not only beneficial for MSCs proliferation and osteogenic differentiation, but also favorable for bone bonding ability on Ti implants in vivo, suggesting that Ti functionalized with CaP and gelatin might have a great potential in clinical joint replacement or dental implants.

Acceleration of de novo bone formation with a novel bioabsorbable film: a histomorphometric study in vivo.

OBJECTIVE: Different types of barrier membranes have been used in periodontal applications for the technology of guided tissue regeneration (GTR). The aim of this study was to characterize the biological effect of novel calcium alginate film (CAF) on bone tissue regeneration by using rabbit mandible defects model. METHODS: A critical size defect (5 mm in diameter) was created in the bilateral corner of mandible of 45 adult rabbits. The defects were covered with CAF served as the experimental group, or conventional collagen membrane (CCM) or left empty as the controls. Animals were killed after 1, 2, 4, 6 and 8 weeks. Morphological and histomorphometric studies were performed to evaluate their bone regeneration pattern and biological effects. RESULTS: Histological sections showed that bone regeneration pattern was centripetal in growth from defect rim. The quantitative histometry analysis revealed a significantly greater percentage of newly generated bone in CAF defects than that in CCM defects and empty defects from 2 to 6 weeks post-operation (P < 0.01). After 6 and 8 weeks, significantly more mature lamella bone had formed with CAF than with CCM. Empty control defects showed bone formation starting from the defect margins and incomplete healing even after 8 weeks. CONCLUSION: The CAF guided early bone growth and appeared more effective as a bioabsorbable GTR membrane than CCM. This study with mandible defect model suggests that bone defects augmented with CAF may offer most promising results from a histological and histomorphometric perspective.

Fabrication and evaluation of Zn containing fluoridated hydroxyapatite layer with Zn release ability.

A biphasic layer with a Zn-containing beta-tricalcium phosphate (ZnTCP) phase and a fluoridated hydroxyapatite (FHA) phase on titanium alloy substrate was prepared by the sol-gel technique. Scanning electron microscopy and energy-dispersive X-ray analysis results showed the ZnTCP/FHA layer to have a heterogeneous surface with microscaled gibbous structures originating from ZnTCP particle agglomeration. This layer had a slow and sustained Zn release behavior. The scratch test result of the ZnTCP/FHA layer was 489+/-4mN, indicating good interface bonding between the layer and substrate. The ZnTCP/FHA layer supported cell growth, and showed a statistically significant increase in cell viability in comparison with another biphasic layer (TCP/FHA) without Zn. This work demonstrates that the present biphasic ZnTCP/FHA layer has the potential to play a significant role in enhancing bone growth when used as the outermost part of bioactive coatings on metallic implants.

Osteochondral repair using the combination of fibroblast growth factor and amorphous calcium phosphate/poly(L-lactic acid) hybrid materials.

A novel amorphous calcium phosphate (ACP)/poly(L-lactic acid) (PLLA) material, which can experience morphological variations in the microstructure is supposed to be a suitable candidate as scaffold for cartilage tissue-engineering. The purpose of this study was to evaluate the efficacy of this scaffold combined with basic fibroblast growth factor (bFGF) to repair articular cartilage defects in a rabbit model. Forty-two osteochondral defects created in the femoral condyles were (a) left untreated, (b) treated by PLLA combined with bFGF, or (c) ACP/PLLA loaded with bFGF. The treatment of PLLA incorporated with bFGF improved defect filling compared with that left untreated, while the regenerated tissue was mainly fibrocartilage and showed little bone formation with only a small amount of collagen type II (Col II) and no aggrecan gene message measured. When implanted with ACP/PLLA and bFGF, most of the defects were filled with a well-established layer of cartilage tissue with abundance of cartilaginous extracellular matrix accumulation observed. Positive immunohistochemical staining of Col II was observed. High levels of Col II and aggrecan message were also detected by RT-PCR. These results indicate the feasibility of using the combination of ACP/PLLA with bFGF for cartilage repair.

Osteoblastic cell response on fluoridated hydroxyapatite coatings.

Fluoridated hydroxyapatite (FHA) coatings were deposited onto Ti6Al4V substrates by sol-gel dip-coating method. X-ray photoelectron spectroscopy results showed that fluoride ions were successfully incorporated into the hydroxyapatite (HA) lattice structure. The dissolution behavior in Tris-buffered physiological saline indicated that all fluoridated HA coatings had lower solubility than that of the pure HA coating. The lowest solubility was obtained at fluoride ion concentrations of 0.8-1.1M. In vitro cell responses were evaluated with human osteosarcoma MG63 cells in terms of cell morphology, proliferation and differentiation (alkaline phosphatase activity and osteocalcin level). For all coatings tested, similar cell morphologies and good cell viability were observed. Coatings fluoridated to 0.8-1.1 had a stronger stimulating effect on cell proliferation and differentiation activities. The influences on cell phenotypes were attributed mainly to a combined ion effect of Ca, P and F released from the coating during dissolution. For the best dissolution resistance and cell activities, it is recommended that the molar level of fluoride ion be from 0.8 to 1.1, such that the coating takes the form of Ca(10)(PO(4))(6)(OH)(1.2-0.9)F(0.8-1.1).

Continuous wave ultrasound enhances vancomycin release and antimicrobial efficacy of antibiotic-loaded acrylic bone cement in vitro and in vivo.

Although antibiotic-loaded bone cement (ALBC) is used as a drug delivery vehicle to decrease infection rates, the varied clinical effect of the antibiotic release remains controversial. The objective of this study is to investigate the enhancement of continuous wave ultrasound (CWU) on vancomycin release and antimicrobial efficacy of ALBC in vitro and in vivo. We measured vancomycin concentrations after a 0.5-h exposure of CWU. The results showed that CWU increased the drug elution by 2.57-27.44% when compared with the controls in vitro. Ultrasonic intensity and vancomycin load both had a significant effect on the cumulative drug elution at 10.5 h, with a significant interaction between each other. We also implanted ALBC specimens into hip joints of sixteen New Zealand White female rabbits after inoculations of Staphylococcus aureus around primary implants for 30 days. Vancomycin concentrations in the hip cavity and urinary elimination of vancomycin were both measured after intermittent exposures of CWU. The results showed that CWU increased local Tmax by 47.6 microg/mL and urinary elimination of vancomycin by 109.56 microg, but failed to prolong local T>MIC. On day 30 after the implantation, assessment in clinical performance, radiology, bacteriology, and histology all showed a tendency of decreased bacterial vitality and relieved inflammation in the infected hip treated by CWU. This study suggested that CWU could effectively enhance vancomycin release and antimicrobial efficacy of ALBC, which may be of clinical significance for treating prosthesis-related infections.

Preparation, characterization and cytocompatibility of porous ACP/PLLA composites.

The purpose of this work was to incorporate amorphous calcium phosphate (ACP) into porous poly(L-lactic acid) (PLLA), because ACP is capable of fast phase transformation and morphological change in body fluid, such, a desired pore wall surface within bone tissue engineering scaffolds can be created. A highly porous ACP/PLLA composite was prepared by a thermally induced phase separation technique. The results showed that the composite had an interconnected pore structure with 100 mum macropores and 10 mum micropores, and 91% porosity; 40 nm primary particles of ACP were agglomerated to 3 mum aggregates, and the aggregates were homogeneously distributed in pore walls; These aggregates showed to be in situ transformed into bone-like apatite after 1 h soaking in phosphate buffered saline solution. Human osteoblast-like cell culture showed that the ACP/PLLA composite had better cell adhesion and alkaline phosphotase activity than pure PLLA. This study demonstrates that the ACP/PLLA composite can enhance cytocompatibility and could act as a promising scaffold for bone tissue engineering.

Porous beta-tricalcium phosphate/collagen composites prepared in an alkaline condition.

Bone substitute materials with natural bone-like structure are considered to be favorable for bone regeneration. In this work, porous beta-tricalcium phosphate (beta-TCP)/collagen composite consisting of bone-like microstructural units was prepared using nanosized beta-TCP particles and alkaline-disassembled collagen. The resulting composite showed a good interconnecting porous structure with approximately 90% porosity and 100 approximately 300 microm pore size. The pore walls were dense, and the combination status of collagen and nanosized beta-TCP particles demonstrated that nanosized beta-TCP particles tightly connected collagen microfibrils as a bone-like microstructural unit. MTT and alkaline phosphatase (ALP) assays showed that the porous composite had enhanced effects on cellular proliferation and activity of osteoblast compared with a control of pure collagen. It is suggested that the adoption of nanosized beta-TCP particles is a main contribution to the formation of the composite with a bone-like microstructural unit, and the unique microstructure could be a main role for the composite to have the positive influence on osteoblast cell proliferation.

In vitro bioactivity and osteoblast-like cell test of zinc containing fluoridated hydroxyapatite films.

Zinc containing fluoridated hydroxyapatite (ZnFHA) films on Ti6Al4V substrates was prepared using sol-gel dip-coating method. The release of zinc ions from ZnFHA film was controlled mainly by the zinc content in the film. The release behavior showed an initial rapid increase release followed by a tapering-off and directed to a constant value at longer time. After soaking in SBF for 8 days, a layer was deposited and completely covered the original surface of the ZnFHA film, indicating good in vitro "bioactivity." The osteoblast-like MG63 cells were seeded on the ZnFHA films; FHA film and Ti6Al4V substrate were used as control. The cell culture result showed that cell adhesion and proliferation on ZnFHA films were significantly increased compared with the controls. The results in this work suggest that ZnFHA films on Ti6Al4V substrates can function as an implant with good bioactivity and cytocompatibility.