Regenerative Efficacy of Supercritical Carbon Dioxide-Derived Bone Graft Putty in Rabbit Bone Defect Model.

Bone defects can arise from numerous reasons, such as aging, tumor, trauma, infection, surgery, and congenital diseases. Bone grafts are commonly used as a substitute to fill the void and regenerate the defect. Due to its clean and green technology, the supercritical carbon dioxide (SCCO2) extraction aided the production of bone grafts is a recent trend. The SCCO2-derived bone graft has osteoconductive and osteoinductive properties along with excellent biocompatible, nontoxic, bioabsorbable, osteoconductive, and good mechanical properties; however, clinical usage during surgery is time-consuming. Therefore, we produced a putty material combining bone graft powder and acellular dermal matrix (ADM) powder and tested its regenerative efficacy in the critical defect in the rabbit model. The putty was found to retain the tubular structure. In addition, the putty depicted excellent stickiness and cohesiveness in both saline and blood medium. The bone regeneration of bone graft and putty was similar; both had excellent bone healing and regeneration of critical defects as evaluated by the X-ray, microtomography, hematoxylin-eosin, Masson trichrome, and alizarin red staining. Putty contains a less washout rate, good mechanical strength, and biocompatibility. In conclusion, the SCCO2-derived moldable putty could be a promising easy-to-use alternative for bone grafts at present which might have real-world usage in orthopedics as a potential bone void filler and dental socket preservation.

Supercritical Carbon Dioxide Decellularized Xenograft-3D CAD/CAM Carved Bone Matrix Personalized for Human Bone Defect Repair.

About 30-50% of oral cancer patients require mandibulectomy and autologous fibula reconstruction. Autograft is the gold standard choice because of its histocompatibility; however, it requires additional surgery from the patient and with possible complications such as loss of fibula leading to calf weakening in the future. Allograft and xenograft are alternatives but are susceptible to immune response. Currently, no personalized bone xenografts are available in the market for large fascial bone defects. In addition, a large-sized complex shape bone graft cannot be produced directly from the raw material. We propose the use of porcine bones with 3D CAD/CAM carving to reconstruct a personalized, wide range and complex-shaped bone. We anticipate that patients can restore their native facial appearance after reconstruction surgery. Supercritical CO2 (SCCO2) technology was employed to remove the cells, fat and non-collagenous materials while maintaining a native collagen scaffold as a biomedical device for bone defects. We successfully developed 3D CAD/CAM carved bone matrices, followed by SCCO2 decellularization of those large-sized bones. A lock-and-key puzzle design was employed to fulfil a wide range of large and complex-shaped maxillofacial defects. To conclude, the 3D CAD/CAM carved bone matrices with lock and key puzzle Lego design were completely decellularized by SCCO2 extraction technology with intact natural collagen scaffold. In addition, the processed bone matrices were tested to show excellent cytocompatibility and mechanical stiffness. Thus, we can overcome the limitation of large size and complex shapes of xenograft availability. In addition, the 3D CAD/CAM carving process can provide personalized tailor-designed decellularized bone grafts for the native appearance for maxillofacial reconstruction surgery for oral cancer patients and trauma patients.

Reconstruction of the orbital floor using supercritical CO2 decellularized porcine bone graft.

Orbital floor fractures subsequently lead to consequences such as diplopia and enophthalmos. The graft materials used in orbital floor fractures varied from autografts to alloplastic grafts, which possess certain limitations. In the present study, a novel porcine bone matrix decellularized by supercritical CO2 (scCO2), ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework. The study was approved by the institutional review board (IRB) of Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUH). Ten cases underwent orbital floor reconstruction in KMUH in 2019. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. Nine out of ten cases used 1 piece of customized ABCcolla® Collagen Bone Graft in each defect. The other case used 2 pieces of customized ABCcolla® Collagen Bone Graft in one defect area due to the curved outline of the defect. In the outpatient clinic, all 10 cases showed improvement of enophthalmos on CT (computerized tomography) at week 8 follow-up. No replacement of implants was needed during follow-ups. To conclude, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate and low complication rate.

Development of a decellularized porcine bone graft by supercritical carbon dioxide extraction technology for bone regeneration.

A series of novel decellularized porcine collagen bone graft (DPB) materials in a variety of shapes and sizes were developed by the supercritical carbon dioxide (SCCO2 ) extraction technique. The complete decellularization of DPB was confirmed by hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining, and residual DNA analysis. The native intact collagen remained in the DPB after the SCCO2 process was confirmed by Masson trichrome staining. The physicochemical characteristics of DPB were investigated by scanning electron microscopy and x-ray diffraction. The cytotoxicity and biocompatibility tests according to ISO10993 and its efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non-toxic. In vitro and in vivo biocompatibility tests of the DPB did not show any toxic or mutagenic effects. The bone regeneration potential of the DPB presented no significant histological differences compared to commercially available deproteinized bovine bone. In conclusion, DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits comparable to that of deproteinized bovine bone. Results from this study could shed light on the potential application of the SCCO2 extraction technique to generate a native decellularized scaffold for bone tissue regeneration in human clinical trials.

Evaluating the bone-regenerative role of the decellularized porcine bone xenograft in a canine extraction socket model.

OBJECTIVE: To evaluate the efficacy of a novel decellularized porcine bone xenograft, produced by supercritical carbon dioxide extraction technology, on alveolar socket healing after tooth extraction compared to a commercially available deproteinized bovine bone (Bio-Oss®). MATERIALS AND METHODS: Nine dogs (about 18 months old and weighing between 20 kg and 30 kg) underwent extractions of lower second to fourth premolars, bilaterally. The dogs were randomly selected and allocated to the following groups: Group 1: control unfilled socket; Group 2: socket filled with decellularized porcine bone xenograft (ABCcolla®) and covered by a commercially available porcine collagen membrane (Bio-Gide®); Group 3: socket filled with Bio-Oss® and covered by Bio-Gide® membrane. One dogs from each group was sacrificed at 4-, 12-, and 24-week to evaluate the socket healing after tooth extraction. The mandible bone blocks were processed without decalcification and specimens were embedded in methyl methacrylate and subjected to histopathology analyses to evaluate the bone regeneration in the extraction sockets. RESULTS: At 24-week after socket healing, ABCcolla® treated defects demonstrated significantly higher histopathology score in new bone formation and bone bridging, but significantly lower score in fluorescent labeling than those of the Bio-Oss®. In the microphotographic examination, decellularized porcine bone xenograft showed similar characteristics of new bone formation to that of Bio-Oss®. However, there was significantly less remnant implant materials in the decellularized porcine bone xenograft compared to the Bio-Oss® group at 24-week. Thus, the decellularized porcine bone graft seems to have promising bone regeneration properties similar to that of Bio-Oss® with less remnant grafted material in a canine tooth extraction socket model. CONCLUSIONS: Within the limits of the study, we concluded that ABCcolla® treated defects demonstrated significantly more new bone formation and better bone bridging, but less amount of fluorescent labeling than those of the Bio-Oss® group. However, clinical studies in humans are recommended to confirm these findings.

Protocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering.

Extracellular matrix (ECM) scaffolds are extensively used in tissue engineering studies and numerous clinical applications for tissue and organ reconstructions. Due to the global severe shortage of human tissues and organs, xenogeneic biomaterials are a common source for human tissue engineering and regenerative medicine applications. Traditional methods for decellularization often disrupt the 3D architecture and damage the structural integrity of the ECM scaffold. To efficiently obtain natural ECM scaffolds from animal tissues and organs with intact architecture, we have developed a platform decellularization process using supercritical CO2 and tested its potential application in tissue engineering. A combination of human mesenchymal stem cells with a decellularized dermal matrix scaffold allowed complete regeneration of skin structure in a porcine full-thickness wound model.

Development of gelatin nanoparticles conjugated with phytohemagglutinin erythroagglutinating loaded with gemcitabine for inducing apoptosis in non-small cell lung cancer cells.

Gelatin is an efficient drug delivery vehicle for attaching targeting molecules like phytohemagglutinin erythroagglutinating (PHA-E) and carrying the chemotherapeutic agent gemcitabine (GEM). Fluorescent gelatin nanoparticles (GNPs) conjugated with PHA-E and carrying gemcitabine (GNP-(PHA-E)-GEM) were synthesized by nanoprecipitation for guiding gemcitabine-loaded gelatin nanoparticles to NSCLC by PHA-E targeting. GNPs have a uniform narrow size distribution and spherical shape, and their particle size is about 290 nm. The release rate of gemcitabine from nanoparticles reached the plateau of the curve at approximately 30% within 72 hours. PHA-E conjugated nanoparticles could enhance the cellular accumulation of nanoparticles. The results showed that GNP-(PHA-E)-GEM treatment caused an increase of cell growth inhibition and cytotoxicity on NSCLC cells A-549 and H292. In an Annexin V/PI assay, treatment with GNP-(PHA-E)-GEM could induce apoptosis of cancer cells. Treatment of NSCLC cells with GNP-(PHA-E)-GEM firstly resulted in time-dependent inhibition of epidermal growth factor receptor (EGFR) and Akt phosphorylation. And it also could increase p53 phosphorylation. And then it could decrease Bad phosphorylation and increase Bax. Finally, it could result in enhancing the release of cytochrome c, which thus increases caspase-9 and caspase-3. In conclusion, GNP-(PHA-E)-GEM could induce growth inhibition and cytotoxicity, which was mediated through inhibition of EGFR phosphorylation and the switching on of p53 that causes cell apoptosis of NSCLC cells A-549 and H292. It's significant to conjugate PHA-E for targeting cancer and inhibiting EGFR phosphorylation as it could decrease the dosage of gemcitabine, which reduces side effects on normal tissue. GNP-(PHA-E)-GEM has great potential for NSCLC treatment.

Fabrication of keratin/fibroin membranes by electrospinning for vascular tissue engineering.

Cardiovascular diseases (CVDs) are some of the leading causes of death and bypass surgery is one of the common treatment options for the critical CVD patients. There is no ideal material available for arterial bypass surgery. Herein, a fibroin and keratin porous membrane was prepared by electro-spinning and proposed for tissue-engineered vascular grafts (TEVGs). The purified fibroin (F) and keratin (K) were mixed in different weight ratios of 9 : 1 (FK91), 8 : 2 (FK82), and 7 : 3 (FK73) to obtain a membrane. The SEM images revealed that the electro-spinned membranes have a fibrous interconnected porous structure. The average diameter of the membrane F, FK91, FK82 and FK73 was 5.74 ± 1.04, 4.20 ± 1.19, 2.94 ± 0.81 and 2.27 ± 0.65 µm, respectively. The ultimate tensile strength (UTS) of F, FK91, FK82 and FK73 was 2.09 ± 0.06, 2.02 ± 0.06, 1.81 ± 0.10 and 1.74 ± 0.12 MPa, respectively. The contact angle of F, FK91, FK82 and FK73 was 72.55 ± 0.55°, 66.39 ± 0.90°, 43.47 ± 0.04° and 33.65 ± 2.83°, respectively. The wettability results were in agreement with those of the cell adhesion to the electro-spinning membranes. The attached HUVECs on the developed membranes showed no cytotoxicity. The immunocytochemistry staining and qPCR analysis showed that the phenotype of the keratin-fibroin membrane was not altered. The results of the ultimate tensile strength, cell adhesion and microstructure revealed that FK82 is similar to native vessels and could be considered as a potential material for TEVGs.

Correction: quantitative analysis of ligand-EGFR interactions: a platform for screening targeting molecules.

[This corrects the article DOI: 10.1371/journal.pone.0116610.].

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Quantitative analysis of ligand-EGFR interactions: a platform for screening targeting molecules.

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in many cancers owing to the binding of ligands such as epidermal growth factor (EGF). Therefore, it is necessary to investigate the interaction between EGFR and its targeting biomolecules. The main aim of this study was to estimate the binding affinity and adhesion force of two targeting molecules, anti-EGFR monoclonal antibody (mAb LA1) and the peptide GE11 (YHWYGYTPQNVI), with respect to EGFR and to compare these values with those obtained for the ligand, EGF. Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (KD) for evaluating the binding affinity. Atomic force microscopy (AFM) was performed to estimate the adhesion force. In the case of EGFR, the KD of EGF, GE11, and mAb LA1 were 1.77 × 10-7, 4.59 × 10-4 and 2.07 × 10-9, respectively, indicating that the binding affinity of mAb LA1 to EGFR was higher than that of EGF, while the binding affinity of GE11 to EGFR was the lowest among the three molecules. The adhesion force between EGFR and mAb LA1 was 210.99 pN, which is higher than that observed for EGF (209.41 pN), while the adhesion force between GE11 and EGFR was the lowest (59.51 pN). These results suggest that mAb LA1 binds to EGFR with higher binding affinity than EGF and GE11. Moreover, the adhesion force between mAb LA1 and EGFR was greater than that observed for EGF and GE11. The SPR and AFM experiments confirmed the interaction between the receptor and targeting molecules. The results of this study might aid the screening of ligands for receptor targeting and drug delivery.

The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation.

To develop a polymer-anticancer drug conjugate, we employed gelatin nanoparticles (GPs) as carriers of cisplatin (CDDP) with anticipated improved therapeutic effect and reduced side effects. The anticancer activities of CDDP-incorporated in GPs (GP-Pt) with biotinylated-EGF (bEGF) modification (GP-Pt-bEGF) were studied. GP-Pt-bEGF with EGFR affinity produced much higher Pt concentrations in A549 cells (high EGFR expression) than in HFL1 cells (low EGFR expression). An in vitro anticancer study showed that GP-Pt-bEGF was more potent than free CDDP or GP-Pt because of its rapid effect on the cell cycle as well as a lower IC(50) (1.2microg/ml) that inhibits A549 cell growth. PI staining showed that cells treated with GP-Pt-bEGF for only 4h had the highest sub-G1 population. The CDDP formulations - free CDDP, GP-Pt, and GP-Pt-bEGF - were given by intratumorous injections to SCID mice in a subcutaneous model. This treatment showed that GP-Pt-bEGF had stronger anti-tumor activity and was less toxic than free CDDP in vivo. Mice treated with GP-Pt-bEGF showed slight body weight loss, whereas free CDDP treatment at the same dose caused a body weight loss of 20-30%. Furthermore, these formulations were given to mice with lung cancer via aerosol delivery. This treatment showed that inhaled GP-Pt-bEGF could target EGFR-overexpressing cells to achieve high cisplatin dosage in cancerous lungs. To summarize, gelatin nanoparticles loaded with CDDP and decorated with EGF tumor-specific ligand were successfully developed. Their in vitro and in vivo targeting ability and anticancer effect were confirmed. The aerosol delivery of the nanodrug carrier was demonstrated. Simple aerosol delivery of targeted drug carriers may prove useful for the clinical treatment of lung cancer patients.