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.

Inhibition of matrix metalloproteinases and inducible nitric oxide synthase by andrographolide in human osteoarthritic chondrocytes.

OBJECTIVE: The aim of this study was to investigate the effects of andrographolide on matrix metalloproteinases (MMP) 1, 3, and 13 and inducible nitric oxide synthase (iNOS) in human articular chondrocytes from osteoarthritic cartilage. METHODS: Passaged chondrocytes were pretreated with or without andrographolide for 2 h, followed by coincubation with interleukin-1 beta (IL-1ß) 1 ng/ml for 24 h. Expression levels of MMP-1, 3, and 13, tissue inhibitor of metalloproteinase-1 (TIMP-1), and iNOS were evaluated using real-time-quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Nitric oxide (NO) was analyzed using the Griess reaction assay. Involvement of nuclear factor kappa B (NF-κB) was assessed by Western blotting, transient transfection, and luciferase reporter assay. RESULTS: Andrographolide tested in these in vitro studies was found be an effective antiarthritic agent, as evidenced by potent inhibition of MMP-1, 3, and 13 and iNOS expression, as well as upregulation of TIMP-1 in IL-1ß-stimulated human articular chondrocytes (p < 0.05). The mechanism of andrographolide's inhibitory effects was mediated by attenuating the activation of NF-κB in human chondrocytes in the presence of IL-1ß. CONCLUSIONS: Andrographolide was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound may merit consideration as a therapeutic agent for treating and preventing osteoarthritis.

Type I collagen scaffold with WNT5A plasmid for in situ cartilage tissue engineering.

BACKGROUND: Cartilage tissue lacks the ability to heal. Cartilage tissue engineering using cell-free scaffolds has been increasingly used in recent years. OBJECTIVE: This study describes the use of a type I collagen scaffold combined with WNT5A plasmid to promote chondrocyte proliferation and differentiation in a rabbit osteochondral defect model. METHODS: Type I collagen was extracted and fabricated into a collagen scaffold. To improve gene transfection efficiency, a cationic chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) vector was used. A solution of TMC/WNT5A complexes was adsorbed to the collagen scaffold to prepare a WNT5A scaffold. Osteochondral defects were created in the femoral condyles of rabbits. The rabbits were divided into defect, scaffold, and scaffold with WNT5A groups. At 6 and 12 weeks after creation of the osteochondral defects, samples were collected from all groups for macroscopic observation and gene expression analysis. RESULTS: Samples from the defect group exhibited incomplete cartilage repair, while those from the scaffold and scaffold with WNT5A groups exhibited "preliminary cartilage" covering the defect. Cartilage regeneration was superior in the scaffold with WNT5A group compared to the scaffold group. Safranin O staining revealed more proteoglycans in the scaffold and scaffold with WNT5A groups compared to the defect group. The expression levels of aggrecan, collagen type II, and SOX9 genes were significantly higher in the scaffold with WNT5A group compared to the other two groups. CONCLUSIONS: Type I collagen scaffold showed effective adsorption and guided the three-dimensional arrangement of stem cells. WNT5A plasmid promoted cartilage repair by stimulating the expression of aggrecan, type II collagen, and SOX9 genes and proteins, as well as inhibiting cartilage hypertrophy.

Restoration of cartilage defects using a superparamagnetic iron oxide-labeled adipose-derived mesenchymal stem cell and TGF-ß3-loaded bilayer PLGA construct.

Aim: We aimed to evaluate the capacity of the bilayer polylactic-co-glycolic acid (PLGA)/TGF-ß3/adipose-derived mesenchymal stem cell (ADSC) construct used to repair cartilage defects and the role of ADSCs in the repair process in vivo. Materials & methods: Defects were created surgically on the femoropatellar groove of knee joints in 64 rabbits. All the rabbits were randomly divided into four groups: defect group, PLGA group, PLGA/TGF-ß3 group and PLGA/TGF-ß3/ADSC group. In vivo MRI and Prussian blue staining were applied. Quantitative real-time PCR and western blot methods were used to analyze the gene and protein expression. Results & conclusion: The result showed that TGF-ß3 could effectively stimulate the expressions of aggrecan, collagen type II and SRY-related HMG box 9 (SOX9). The bilayer PLGA/TGF-ß3/ADSC construct showed a promising repair effect.

Knockdown of KIAA1199 suppresses IL-1ß-induced cartilage degradation and inflammatory responses in human chondrocytes through the Wnt/ß-catenin signalling pathway.

The overproduction of proteolytic enzymes and dysregulation of extracellular matrix (ECM) metabolism have been shown to accelerate the degradation process of articular cartilage. The purpose of this study was to investigate the role of KIAA1199 and its association with the pathophysiology of osteoarthritis (OA). We found that the expression of KIAA1199 was significantly upregulated in OA cartilage compared with normal tissues. Serum levels of KIAA1199 were higher in OA patients than in non-OA patients. Furthermore, knockdown of KIAA1199 inhibited interleukin-1 beta (IL-1ß)-induced ECM metabolic imbalance by regulating the expression of A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5; matrix metallopeptidase-13; aggrecan; and COL2A1. In addition, silencing of KIAA1199 significantly decreased the expression of inflammatory mediators such as prostaglandin E2, IL-6, and TNF-α. Mechanistic analyses further revealed that IL-1ß-induced activation of the Wnt/ß-catenin pathway was suppressed during KIAA1199 knockdown. Moreover, KIAA1199 expression was also upregulated in an in vivo rat OA model. Together, these results increase our understanding of the emerging role of KIAA1199 in the process of OA degeneration, and may lead to a novel molecular target to prevent cartilage degradation.

Ligament regeneration using a knitted silk scaffold combined with collagen matrix.

This study was aimed to develop a new practical ligament scaffold by synergistic incorporation of silk fibers, a knitted structure, and a collagen matrix. The efficacy for ligament tissue engineering was investigated in vitro and in animal models. Cells cultured on a collagen substrate expressed ligament matrix genes at higher levels than those on a silk substrate. The silk scaffold elicited little inflammatory reaction and degraded slowly after subcutaneous implantation in a mouse model. In the rabbit MCL defect model, MCLs treated with a silk+collagen scaffold deposited more collagen, had better mechanical properties, and showed more native microstructure with larger diameter collagen fibrils and stronger scaffold-ligament interface healing than untreated MCLs and those treated with silk scaffolds. These results demonstrated that the knitted silk+collagen sponge scaffold improves structural and functional ligament repair by regulating ligament matrix gene expression and collagen fibril assembly. The findings are the first to highlight the important roles of biomaterials in ligament regeneration biology. Also, the concept of an "internal-space-preservation" scaffold is proposed for the tissue repair under physical loading.

Successful endoscopic removal of three embedded esophageal self-expanding metal stents.

In the report, we describe a case of refractory benign esophageal strictures from esophageal cancer after an operation for the placement of three partially covered self-expanding metal stents (SEMSs), which were all embedded in the esophageal wall. Using the stent-in-stent technique, the three embedded SEMSs were successfully removed without significant complications. To the best of our knowledge, few cases of the successful removal of multiple embedded esophageal SEMSs have been reported in the literature. This case also highlights that the stent-in-stent technique is effective for removing multiple embedded esophageal SEMSs.

Reconstruction of segmental bone defects in the rabbit ulna using periosteum encapsulated mesenchymal stem cells-loaded poly (lactic-co-glycolic acid) scaffolds.

BACKGROUND: Repair of large bone defects remains a challenge for clinicians. The present study investigated the ability of mesenchymal stem cells (MSCs) and/or periosteum-loaded poly (lactic-co-glycolic acid) (PLGA) to promote new bone formation within rabbit ulnar segmental bone defects. METHODS: Rabbit bone marrow-derived MSCs (passage 3) were seeded onto porous PLGA scaffolds. Forty segmental bone defects, each 15 mm in length, were created in the rabbit ulna, from which periosteum was obtained. Bone defects were treated with either PLGA alone (group A), PLGA + MSCs (group B), periosteum-wrapped PLGA (group C) or periosteum-wrapped PLGA/MSCs (group D). At 6 and 12 weeks post-surgery, samples were detected by gross observation, radiological examination (X-ray and micro-CT) and histological analyses. RESULTS: Group D, comprising both periosteum and MSCs, showed better bone quality, higher X-ray scores and a greater amount of bone volume compared with the other three groups at each time point (P < 0.05). No significant differences in radiological scores and amount of bone volume were found between groups B and C (P > 0.05), both of which were significantly higher than group A (P < 0.05). CONCLUSIONS: Implanted MSCs combined with periosteum have a synergistic effect on segmental bone regeneration and that periosteum plays a critical role in the process. Fabrication of angiogenic and osteogenic cellular constructs or tissue-engineered periosteum will have broad applications in bone tissue engineering.

Cell transplantation for articular cartilage defects: principles of past, present, and future practice.

As articular cartilage has very limited self-repair capability, the repair and regeneration of damaged cartilage is a major challenge. This review aims to outline the past, present, and future of cell therapies for articular cartilage defect repair. Autologous chondrocyte implantation (ACI) has been used clinically for more than 20 years, and the short, medium, and long-term clinical outcomes of three generation of ACI are extensively overviewed. Also, strategies of clinical outcome evaluation, ACI limitations, and the comparison of ACI clinical outcomes with those of other surgical techniques are discussed. Moreover, mesenchymal stem cells and pluripotent stem cells for cartilage regeneration in vitro, in vivo, and in a few clinical studies are reviewed. This review not only comprehensively analyzes the ACI clinical data but also considers the findings from state-of-the-art stem cell research on cartilage repair from bench and bedside. The conclusion provides clues for the future development of strategies for cartilage regeneration.

Electrospun nanofibrous matrix improves the regeneration of dense cortical bone.

Numerous in vitro studies have indicated the potential of using electrospun nanofibrous scaffolds for tissue regeneration. However, few reports have demonstrated their utility in real tissue repair models. The present investigation tested the hypothesis that electrospun poly-L-lactic acid (PLLA) nanofibrous membrane leads to dense cortical bone regeneration and improves the efficacy of currently-used collagenous guided bone regeneration (GBR) membrane. In vitro, the function of bone marrow-derived mesenchymal stem cells (BMSCs) on nanofibrous scaffolds was evaluated. In an in vivo experiment, large bony defects were created in rabbit tibia and treated with a nanofiber-reinforced bilayer membrane, nanofibrous membrane, or collagenous membrane alone. Three and six weeks after operation, bone defect healing was assessed radiologically and histologically. In vitro differentiation studies showed that BMSCs had much higher expression of Runx2 and collagen type I, alpha 1 mRNAs, when cultured on nanofibrous scaffolds. The radiographic and histological data both showed that the group treated with bilayer membrane had more bony tissue formation at 3 weeks. Moreover, at 6 weeks, only the bilayer membrane-treated bone defects displayed better regeneration of cortical bone tissue, whereas in the other groups the defects were filled with spongy bone-like tissue. The results demonstrated that electrospun nanofibrous membrane improves the regeneration of cortical bone, suggesting that this type of membrane can be combined with current collagenous GBR membrane to improve guided bone regeneration technology.

The inductive effect of bone morphogenetic protein-4 on chondral-lineage differentiation and in situ cartilage repair.

OBJECTIVES: As recent studies have suggested that bone morphogenetic protein-4 (BMP-4) and BMP-7 are promising cartilage differentiation factors, this study aimed to compare the efficacy of BMP-4 and BMP-7 for chondral-lineage differentiation in vitro as well as the efficacy of BMP-4 for articular cartilage repair in vivo. METHODS: Rabbit mesenchymal stromal cells and articular chondrocytes were treated with 10 ng/mL human recombinant BMP-4 or BMP-7. The expression of cartilage-specific genes (col II, aggrecan, and Sox9) and fibroblast growth factor receptor genes was tested by real-time polymerase chain reaction in vitro. Also, full-thickness cartilage defects (diameter 4 mm, thickness 3 mm) were created in New Zealand white rabbits and untreated (group I), or treated with a bilayer collagen scaffold (group II) or BMP-4 with scaffold (group III) (n = 12/group). The repaired tissues were harvested for histology and mechanical testing after 6 or 12 weeks. RESULTS: Cartilage differentiation of mesenchymal stromal cells was more apparent after BMP-4 treatment, as evidenced by higher expression of type II collagen and aggrecan genes. Also, BMP-4 induced higher aggrecan and fibroblast growth factor receptor-2 gene expression in chondrocytes, whereas BMP-7 had no effect. In the in vivo experiments, group III treated with BMP-4 protein had the largest amounts of cartilage tissue, which restored a greater surface area of the defect and achieved higher International Cartilage Repair Society scores. Moreover, Young's modulus, which indicates the mechanical properties of the repaired tissue, was markedly higher in group III than in groups I and II (p < 0.05), but lower than in normal tissue. CONCLUSION: BMP-4 is more potent than BMP-7 for cartilage differentiation. The delivery of BMP-4 protein in a bilayer collagen scaffold stimulates the formation of cartilage tissue.

Local delivery of autologous platelet in collagen matrix simulated in situ articular cartilage repair.

Bone marrow released by microfracture or full-thickness cartilage defect can initiate the in situ cartilage repair. However, it can only repair small cartilage defects (<2 cm(2)). This study aimed to investigate whether autologous platelet-rich plasma (PRP) transplantation in collagen matrix can improve the in situ bone marrow-initiated cartilage repair. Full-thickness cartilage defects (diameter 4 mm, thickness 3 mm) in the patellar grooves of male New Zealand White rabbits were chosen as a model of in situ cartilage repair. They were treated with bilayer collagen scaffold (group II), PRP and bilayer collagen scaffold (group III), and untreated (group I), respectively (n = 11). The rabbits were sacrificed at 6 and 12 weeks after operation. The repaired tissues were processed for histology and for mechanical test. The results showed that at both 6 and 12 weeks, group III had the largest amounts of cartilage tissue, which restored a larger surface area of the cartilage defects. Moreover, group III had higher histological scores and more glycosaminoglycans (GAGs) content than those in the other two groups (p < 0.05). The Young's modulus of the repaired tissue in group II and group III was higher than that of group I (p < 0.05). Autologous PRP and bilayer collagen matrix stimulated the formation of cartilage tissues. The findings implicated that the combination of PRP with collagen matrix may repair larger cartilage defects that currently require complex autologous chondrocyte implantation (ACI) or osteochondral grafting.