Bone Regeneration Using Duck's Feet-Derived Collagen Scaffold as an Alternative Collagen Source.

Collagen is an important component that makes 25-35% of our body proteins. Over the past decades, tissue engineers have been designing collagen-based biocompatible materials and studying their applications in different fields. Collagen obtained from cattle and pigs has been mainly used until now, but collagen derived from fish and other livestock has attracted more attention since the outbreak of mad cow disease, and they are also used as a raw material for cosmetics and foods. Due to the zoonotic infection using collagen derived from pigs and cattle, their application in developing biomaterials is limited; hence, the development of new animal-derived collagen is required. In addition, there is a religion (Islam, Hinduism, and Judaism) limited to export raw materials and products derived from cattle and pig. Hence, high-value collagen that is universally accessible in the world market is required. Therefore, in this review, we have dealt with the use of duck's feet-derived collagen (DC) as an emerging alternative to solve this problem and also presenting few original investigated bone regeneration results performed using DC.

The Cocktail Effect of BMP-2 and TGF-ß1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model.

Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-ß1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-ß1 (10 ng) (GC/BMP-2/TGF-ß1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-ß1 (5 ng) (GC/BMP-2/TGF-ß1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-ß1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.

Supramolecular Hydrogels Based on MPEG-Grafted Hyaluronic Acid and α-CD Containing HP-ß-CD/Simvastatin Enhance Osteogenesis In Vivo.

Simvastatin (SIM) accelerates new bone formation both in vitro and In Vivo by enhancing the expression of recombinant human bone morphogenetic protein-2 (rhBMP-2). In this study, we evaluated the effect of water-solubility of SIM on new bone formation by preparing two types of supramolecular hydrogels: pseudopolyrotaxanes (PPRXs) based on metoxy polyethyleneglycol-grafted hyaluronic acid (MPEG-g-HA) and α-cyclodextrin (α-CD) containing water-soluble hydroxypropyl ß-cyclodextrin/simvastatin inclusion complex (HP-ß-CD-ic-SIM; MPEG-g-HA/α-CD/HP-ß-CD-ic-SIM) or only SIM (MPEG-g-HA/α-CD/SIM). As compared to MPEG-g- HA/α-CD/SIM, SIM was more rapidly released from MPEG-g-HA/α-CD/HP-ß-CD-ic-SIM in a sustained manner owing to increased water-solubility. New bone actively formed at the calvarial defect site in a rabbit model 4 weeks after implantation, as examined by micro computed tomography (micro CT), hematoxylin and eosin (H&E) staining, and Goldner's trichrome staining. The results showed that the water-solubility of SIM plays a significant role in enhancing new bone formation in vivo.

Preparation and Evaluation of Dexamethasone (DEX)/Growth and Differentiation Factor-5 (GDF-5) Surface-Modified Titanium Using ß-Cyclodextrin-Conjugated Heparin (CD-Hep) for Enhanced Osteogenic Activity In Vitro and In Vivo.

The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti) samples of disc and screw types using dexamethasone (DEX) and/or growth and differentiation factor-5 (GDF-5), as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8) assay, as well as the measurements of alkaline phosphatase (ALP) activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5-Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5-Ti and host bone, as evaluated by micro computed-tomography (micro CT) analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

Effects of Titanium Mesh Surfaces-Coated with Hydroxyapatite/ß-Tricalcium Phosphate Nanotubes on Acetabular Bone Defects in Rabbits.

The management of severe acetabular bone defects in revision reconstructive orthopedic surgery is challenging. In this study, cyclic precalcification (CP) treatment was used on both nanotube-surface Ti-mesh and a bone graft substitute for the acetabular defect model, and its effects were assessed in vitro and in vivo. Nanotube-Ti mesh coated with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) was manufactured by an anodizing and a sintering method, respectively. An 8 mm diameter defect was created on each acetabulum of eight rabbits, then treated by grafting materials and covered by Ti meshes. At four and eight weeks, postoperatively, biopsies were performed for histomorphometric analyses. The newly-formed bone layers under cyclic precalcified anodized Ti (CP-AT) meshes were superior with regard to the mineralized area at both four and eight weeks, as compared with that under untreated Ti meshes. Active bone regeneration at 2-4 weeks was stronger than at 6-8 weeks, particularly with treated biphasic ceramic (p < 0.05). CP improved the bioactivity of Ti meshes and biphasic grafting materials. Moreover, the precalcified nanotubular Ti meshes could enhance early contact bone formation on the mesh and, therefore, may reduce the collapse of Ti meshes into the defect, increasing the sufficiency of acetabular reconstruction. Finally, cyclic precalcification did not affect bone regeneration by biphasic grafting materials in vivo.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Nano-Layered Growth and Differentiation Factor-5.

Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation.

Accelerating bone regeneration using poly(lactic-co-glycolic acid)/hydroxyapatite scaffolds containing duck feet-derived collagen.

Collagen, with low antigenicity and excellent cell adhesion, is a biomaterial mainly used for regenerating bone, cartilage, and skin, owing to its biocompatibility and biodegradability. Results from a previous study confirmed that a scaffold mixed with duck feet-derived collagen (DC) and Poly(lactic-co-glycolic acid) (PLGA) reduced inflammatory reaction and increased bone regeneration. To develop an optimal bone substitute we included hydroxyapatite (HAp), a key osteoconductive material, in a DC and PLGA mixture. We fabricated 0, 10, 20, 40, 60, and 80 wt% DC/PLGA/HAp scaffolds and studied their potential for bone tissue engineering. Characteristic analysis of the scaffold and seeding of rabbit bone marrow mesenchymal stem cells (rBMSCs) on the scaffold were conducted to investigate cell proliferation, osteogenic differentiation, and bone formation. We confirmed that increasing DC concentration not only improved the compressive strength of the DC/PLGA/HAp scaffold but also cell proliferation and osteogenic differentiation. It was found through comparison with previous studies that including HAp in the scaffold also promotes osteogenic differentiation. Our study thus shows through in vivo results that the 80 wt% DC/PLGA/HAp scaffold promotes bone mineralization and collagen deposition while reducing the inflammatory response. Hence, 80 wt% DC/PLGA/HAp has excellent potential as a biomaterial for bone regeneration applications.

Biomimetic sponge using duck's feet derived collagen and hydroxyapatite to promote bone regeneration.

Collagen, a natural biomaterial derived from animal tissues, has attracted the attention of biomedical material researchers because of its excellent cell affinity and low rejection in vivo. In this study, collagen was extracted using livestock by-product flippers, and an experiment was performed to assess its application as a scaffold for bone tissue implantation. For this purpose, we fabricated 2%, and 3% duck's feet derived collagen (DC) sponges. We then compared them to hydroxyapatite (HAp)-coated DC sponges, and measured the porosity and pore size using scanning electron microscopy (SEM) to analyze the physical properties and morphology of DC and DC/HAp sponges. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were carried out to measure the proliferation of bone marrow stem cells (BMSCs) in DC and DC/HAp sponges. An alkaline phosphatase activity assay confirmed the osteogenic differentiation ability of BMSCs. Polymerase chain reaction (PCR) was performed to confirm the BMSC-specific genetic marker. The osteogenic potential was confirmed by the bone formation in an in vivo environment on the scaffold by histological and immunohistochemical analysis. Overall, this study shows that DC/HAp sponges have biocompatibility and good physical properties. Additionally, DC/HAp sponges show potential use as bone graft materials for tissue engineering applications.

Repair of diaphyseal bone defects with calcitriol-loaded PLGA scaffolds and marrow stromal cells.

Calcitriol (1,25(OH)2D3)-loaded porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds prepared by solvent casting/salt leaching method were used to repair a 1.5 cm diaphyseal segmental bone defect as a fully absorbable osteogenic biomaterial. The in vitro release of sulforhodamine B (SRB) from PLGA scaffold was measured using spectrophotometer, considering SRB as a model drug. The SRB released from SRB-incorporated PLGA scaffold during 3 months was with relatively low initial burst. The calcitriol-loaded PLGA scaffolds with or without marrow stromal cells (MSCs) were implanted in a critical-sized intercalated bone defect in rabbit femur. Defects were assessed by radiographs until 9 weeks. The bony union of the defect was observed only in the calcitriol-loaded groups. RT-PCR results indicated that MSCs, which were seeded into calcitriol-loaded scaffold, expressed an increased level of alkaline phosphatase, osteonectin, and type I collagen mRNA at day 10. After 2 and 4 weeks, the implanted scaffolds were evaluated by histology. New osteoid matrix and direct calcium deposits were more evident in calcitriol/PLGA/MSC group. Three-dimensional computed tomography and frontal tomographic images of repaired femur showed that normal femur anatomy had been restored with cortical bone with no implanted PLGA remnants at 20 weeks. It can be concluded that the porous calcitriol-loaded PLGA scaffold combined with MSCs may be a novel method for repairing the large loaded bone defect.

Outcomes of polyethylene liner cementation into a fixed metal acetabular shell with minimum follow-up of 7 years.

Cementation of a polyethylene liner into the well-fixed shell is a convenient option for revision total hip arthroplasty. We retrospectively reviewed 45 patients who had liner cementation to investigate the risk factors which gave rise to major complications and reoperation. Patients were observed for a minimum of 7 years (range 7.8-14 years). Relevant risk factors (age, BMI, surgical approach, previous cup size and position, types of coated surface) were assessed with Cox regression analysis. The mean Harris Hip Score was improved from 62.5 (range 57-68) preoperatively to 87.1 (range 70-97). A total of 7 hips (15.5%) had acetabular component loosening that was treated with reoperation. Prevalence of acetabular component loosening was statistically significantly higher in hydroxyapatite-coated group (5 of 13) than in the Ti-coated group (2 of 32, p = 0.015). All recurrent dislocations occurred in patients treated with a posterior approach. Diameter of the previous metal shell of below 54 mm showed a lower 10-year survival rate than those greater than 54 mm in diameter. PE liner cementation in stable metal cup is a useful alternative option for carefully selected patients. Pre-existing HA-coated cups as well as small sized cups were indicative of poor outcomes.

Enhancement of tibial regeneration in a rat model by adipose-derived stromal cells in a PLGA scaffold.

INTRODUCTION: Autologous adipose-derived stromal cells (ASCs) are an obvious source of osteogenic cells and can be easily isolated from adipose tissue. We evaluated the potential of ASCs seeded onto a scaffold to heal tibial defects. METHODS: Autologous ASCs were obtained from adipose tissue by collagenase digestion. The cells were seeded in three-dimensional poly(lactic)-glycolic acid (PLGA) scaffolds and cultured in osteogenic medium for four weeks. Evidence of osteogenesis was assessed by von Kossa staining in three-dimensional cultures following osteogenic induction. The critical size tibial defects (10mm) were created using a rat model. Defects were either left empty (sham group), treated with a PLGA scaffold alone (PLGA group), or a PLGA/ASC composite (PLGA/ASC group). Using radiologic and histologic analyses, we assessed total bone volume and vascular density. Total RNA was prepared from regenerated bone and analyzed for osteogenic marker gene expression. RESULTS: In three-dimensional cultures, the PLGA/ASC composite showed multiple calcified extracellular matrix nodules on von Kossa staining after four weeks of differentiation. Near complete healing was observed between the PLGA/ASC engrafted tibial defects on plain radiographs and micro-CT findings. Total bone volume and mechanical strength were significantly higher in the PLGA/ASC group compared to the sham and PLGA groups. Histologic analysis revealed increased new bone formation along capillaries in the PLGA/ASC group. Real-time RT-PCR analysis revealed a significant increase in the expression of osteogenic genes in the PLGA/ASC group. CONCLUSIONS: The results showed that the repair of tibial defects was accelerated by implantation of autologous ASCs seeded onto a PLGA scaffold. Therefore, PLGA/ASC is a promising new cell-based therapy for healing critical size tibial defects.

Eleven-year follow-up of second-generation metal-on-metal total hip arthroplasty.

PURPOSE: To investigate the cause of failure in matte-surface cemented stems in second-generation metal-on- metal total hip arthroplasty (THA). METHODS: Records of 26 men and 11 women (39 hips) aged 29 to 72 years who underwent primary cementless THAs by a single surgeon using second-generation metal-on-metal prostheses and were followed up for a mean of 122 (range, 120-141) months were reviewed. Two types of femoral stems were used: a cementless Ti-alloy stem (n=21) and a matte-surface, iron-based alloy, cemented stem (n=18). Clinical outcomes were measured using the Harris hip score. Radiographs were assessed for stem loosening and osteolysis. Patient activity levels were graded. Surfaces of the retrieved femoral stems and periprosthetic tissue samples were examined. Metallic and cement particles were studied. Hypersensitivity to metal was tested. RESULTS: None of the cementless stems were revised; no osteolysis or stem loosening occurred. In contrast, 7 of 18 matte-surface cemented stems were revised owing to stem loosening or osteolysis. Periprosthetic tissues revealed abundant cement-related particles; 90% were zirconium oxides but a few were iron particles. Histological examination of periprosthetic tissues showed perivascular infiltration of lymphocytes and macrophages containing tiny foreign materials. Metal hypersensitivity was not associated with aseptic loosening. CONCLUSION: Metal-on-metal THA with cementless components could be recommended for young, active patients to prevent wear and osteolysis. The matte-surface cemented stem is more likely to fail owing to friction during the earlier stage and cement-related biological processes during the later stage.

Reduction of inflammatory reaction of poly(d,l-lactic-co-glycolic Acid) using demineralized bone particles.

Poly(lactide-co-glycolic acid) (PLGA) has been widely applied to tissue engineering as a good biocompatible material because of its biodegradability and nontoxic metabolites, but how the inflammatory reaction of PLGA on the surrounding tissue in vivo is reduced has not been discussed sufficiently. We hypothesized that the cells neighboring the PLGA implant might have an inflammatory response that could be reduced by impregnating demineralized bone particles (DBPs) into the PLGA. We manufactured five different ratios of DBP/PLGA hybrid materials, with each material containing 0, 10, 20, 40, and 80 wt% of DBPs of PLGA. For biocompatibility test, NIH/3T3 mouse fibroblasts were cultured on the DBP/PLGA scaffold for 3 days. The inflammatory potential of PLGA was evaluated using messenger ribonucleic acid expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) on a human acute promyelocytic leukemic cell (HL-60). The in vivo response of DBP/PLGA film was compared with that of PLGA film implanted subcutaneously; the local inflammatory response was observed according to histology. The DBP/PLGA scaffold had no adverse effect on NIH/3T3 initial cell attachment and did not affect cell viability. DBP/PLGA films, especially PLGA films containing 80% DBP, elicited a significantly lower expression of IL-1beta and TNF-alpha from HL-60 cells than PLGA film alone. In vivo, DBP/PLGA film demonstrated a more favorable tissue response profile than PLGA film, with significantly less inflammation and fibrous capsule formation as below only 20% of DBP in PLGA film during implantation. This study shows that application of DBPs reduces the fibrous tissue encapsulation and foreign body giant cell response that commonly occurs at the interface of PLGA.

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold.

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.

Correlation of proliferation, morphology and biological responses of fibroblasts on LDPE with different surface wettability.

In order to find a correlation between cell adhesion, growth and biological response with different wettability, NIH/3T3 fibroblast cells were cultured on plasma-treated low-density polyethylene (LDPE) film generated with radio frequency. Different surface wettabilities (water contact angle 90-40 degrees ) were created by varying the duration of plasma treatment between 0 and 15 s, respectively. Growth and proliferation rate of cells on LDPE surfaces was evaluated by MTT assay, and cell morphology, by means of spreading and adhesion, was characterized by scanning electron microscopy (SEM). The expression of particular genes in cells contacted on films with different wettability was analyzed by RT-PCR. Using the MTT assay, we confirmed that the amount of cell adhesion was higher on surface of film with a water contact angle of 60 degrees than with other water contact angle. Also, the proliferation rate of cells was highest with a water contact angle of 60 degrees . It was confirmed by SEM that the morphology of cells adhered with a water contact angle of 50-60 degrees was more flattened and activated than on other surfaces. Furthermore, c-fos mRNA in cells showed maximum expression on the film with contact angle range of 50-60 degrees and c-myc mRNA expressed highly on the film with a contact angle of 50 degrees . Finally, p53 gene expression increased as wettability increase. These results indicate that a water contact angle of the polymer surfaces of 50-60 degrees was suitable for cell adhesion and growth, as well as biological responses, and the surface properties play an important role for the morphology of adhesion, growth and differentiation of cells.