ABSTRACT
BACKGROUND: Antibiotic-containing carrier systems are one option that offers the advantage of releasing active ingredients over a longer period of time. In vitro sustained drug release from a carrier system consisting of microporous ß-TCP ceramic and alginate has been reported in previous works. Alginate dialdehyde (ADA) gelatin gel showed both better mechanical properties when loaded into a ß-TCP ceramic and higher biodegradability than pure alginate. METHODS: Dual release of daptomycin and BMP-2 was measured on days 1, 2, 3, 6, 9, 14, 21, and 28 by HPLC and ELISA. After release, the microbial efficacy of the daptomycin was verified and the biocompatibility of the composite was tested in cell culture. RESULTS: Daptomycin and the model compound FITC protein A (n = 30) were released from the composite over 28 days. A Daptomycin release above the minimum inhibitory concentration (MIC) by day 9 and a burst release of 71.7 ± 5.9% were observed in the loaded ceramics. Low concentrations of BMP-2 were released from the loaded ceramics over 28 days.
Subject(s)
Anti-Bacterial Agents , Bone Morphogenetic Protein 2 , Calcium Phosphates , Ceramics , Daptomycin , Gelatin , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/metabolism , Daptomycin/chemistry , Daptomycin/pharmacology , Gelatin/chemistry , Ceramics/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Calcium Phosphates/chemistry , Animals , Microbial Sensitivity Tests , Mice , Drug Carriers/chemistry , Drug LiberationABSTRACT
Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (ß-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.
Subject(s)
Osteogenesis , Tissue Scaffolds , Calcium Phosphates/pharmacology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Hydrodynamics , Tissue Engineering/methods , Tissue Scaffolds/chemistryABSTRACT
Rapid and efficient vascularization is still considerably challenging for a porous ß-tricalcium phosphate (ß-TCP) scaffold to achieve. To overcome this challenge, branched channels were created in the porous ß-TCP scaffold by using 3D printing and a template-casting method to facilitate the instant flow of blood supply. Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were seeded in the channeled porous scaffolds and characterized through a double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay, and cell migration. Channeled porous ß-TCP scaffolds were then implanted in the subcutaneous pockets of mice. Histological staining and immunohistochemical staining on vascularization and bone-related markers were carried out on the embedded paraffin sections. Results from in vitro experiments showed that branched channels significantly promoted HUVECs' infiltration, migration, proliferation, and angiogenesis, and also promoted the proliferation and osteogenesis differentiation of hBMSCs. In vivo implantation results showed that, in the early stage after implantation, cells significantly migrated into branched channeled scaffolds. More matured blood vessels formed in the branched channeled scaffolds compared to that in nonchanneled and straight channeled scaffolds. Beside promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous ß-TCP scaffold promoted rapid vascularization and potentially stimulated bone cells recruitment.
Subject(s)
Tissue Engineering , Tissue Scaffolds , Mice , Humans , Animals , Tissue Scaffolds/chemistry , Tissue Engineering/methods , Porosity , Neovascularization, Physiologic , Calcium Phosphates/chemistry , Osteogenesis , Human Umbilical Vein Endothelial Cells , Neovascularization, PathologicABSTRACT
The extracellular matrix microenvironment, including chemical constituents and topological structure, plays key role in regulating the cell behavior, such as adhesion, proliferation, differentiation, apoptosis, etc. Until now, to investigate the relationship between surface texture and cell response, various ordered patterns have been prepared on the surface of different matrixes, whereas almost all these strategies depend on advanced instruments or severe synthesis conditions. Herein, cell-mediated mineralization method has been applied to construct nanopattern on the surface of ß-TCP scaffold. The formation process, morphology, and composition of the final pattern were characterized, and a possible mineralization mechanism has been proposed. Moreover, the cell behavior on the nanopattern has been investigated, and the results showed that the mouse bone marrow mesenchyme stem cells (mBMSCs) display good affinity with the nanopattern, which was manifested by the good proliferation and osteogenic differentiation status of cells. The synthetic strategy may shed light to construct advanced topological structures on other matrixes for bone repair.
Subject(s)
Mesenchymal Stem Cells , Osteogenesis , Animals , Cell Differentiation , Extracellular Matrix , MiceABSTRACT
Aim: The objectives of this study were to develop a new decellularized bone matrix (DBM) and to investigate its effect on the in vitro cell behavior of human bone marrow-derived mesenchymal stem cells (hMSCs), compared with porous ß-tricalcium phosphate (ß-TCP) scaffolds. Materials & methods: Triton X-100 and deoxycholate sodium solution, combining DNase I and RNase, were used to decellularize porcine bones. The DBM were then characterized by DNA contents and matrix components. hMSCs were then seeded on the DBM and ß-TCP scaffolds to study cell behavior. Results: Results showed that most porcine cells were removed and the matrix components of the DBM were maintained. Cell culture results showed that DBM promoted cell attachment and proliferation of hMSCs but did not significantly promote the gene expression of osteogenic genes, compared with ß-TCP scaffolds. Conclusion: DBM has similar function on cell behavior to ß-TCP scaffolds that have promising potential in bone tissue regeneration.
Subject(s)
Bone Matrix/cytology , Bone Regeneration , Extracellular Matrix/chemistry , Mesenchymal Stem Cells/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , SwineABSTRACT
Inadequate oxygen and nutrient diffusion in a porous scaffold often resulted in insufficient formation of branched vasculatures, which hindered bone regeneration. In this study, interconnected porous ß-tricalcium phosphate (ß-TCP) scaffolds with different geometric designs of channels were fabricated and compared to discover the functionality of structure on facilitating nutrient diffusion for angiogenesis. In vitro fluid transportation and degradation of the scaffolds were performed. Cell infiltration, migration, and proliferation of human umbilical vein endothelial cells (HUVECs) on the scaffolds were carried out under both static and dynamic culture conditions. A computational simulation model and a series of immunofluorescent staining were implemented to understand the mechanism of cell behavior in response to different types of scaffolds. Results showed that geometry with multiple channels significantly accelerated the release of Ca2+ and increased the fluid diffusion efficiency. Moreover, multiple channels promoted HUVECs' infiltration and migration in vitro. The ex vivo implantation results showed that the channels promoted cells from the rats' calvarial bone explants to infiltrate into the implanted scaffold. Multiple channels also stimulated HUVECs' proliferation prominently at both static and dynamic culturing conditions. The expression of both cell migration-related protein α5 and angiogenesis-related protein CD31 on multiple-channeled scaffolds was upregulated compared to that on the other two types of scaffolds, implying that multiple channels reinforced cell migration and angiogenesis. All the findings suggested that the geometric design of multiple channels in the porous ß-TCP scaffold has promising potential to promote cell infiltration, migration, and further vascularization when implanted in vivo.
Subject(s)
Calcium Phosphates/chemistry , Cell Proliferation , Neovascularization, Physiologic , Tissue Scaffolds/chemistry , Cell Adhesion , Cell Movement , Human Umbilical Vein Endothelial Cells , Humans , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Porosity , Tissue EngineeringABSTRACT
The repair of craniofacial bony defects by traditional grafting techniques requires substantial time and effort, with associated morbidity. Tissue engineering has therefore become a novel approach targeting application for bone regeneration. This study used the rabbit model for radiographic and histological evaluation of bone bioengineering for mandibular defects reconstruction using only ß-tricalcium phosphate (ß-TCP) and, when loaded with autogenous; bone marrow-derived undifferentiated mesenchymal stem cells (BM-MSCs). Critical-sized defects (10 × 15 mm) were created unilaterally in the mandibular body region of each rabbit (n = 16), to be filled with the BM-MSCs/ß-TCP constructs for the study group (group I) (n1 = 8) and with scaffold devoid of cells for the control group (group II) (n2 = 8). Two rabbits from each group were sacrificed after healing periods of 2, 4, 12, and 24 weeks. The results revealed that the BM-MSCs endowed ß-TCP scaffold with a better and more rapid bone regenerating potential: since the first evaluation period of 2 weeks, the regenerated bone tissue in group I was more mature, denser and homogeneously distributed. From these findings we could infer that the bone regeneration process was jump-started within the study group cases, which led to better quality of regenerated bone.