β-TCP Scaffolds Research Articles

The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and β-TCP scaffold

Extracellular matrix (ECM) serves a key role in cell migration, attachment, and cell development. Here we report that ECM derived from human umbilical vein endothelial cells (HUVEC) promoted osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSC). We first produced an HUVEC-derived ECM on a three-dimensional (3D) beta-tricalcium phosphate (β-TCP) scaffold by HUVEC seeding, incubation, and decellularization. The HUVEC-derived ECM was then characterized by SEM, FTIR, XPS, and immunofluorescence staining. The effect of HUVEC-derived ECM-containing β-TCP scaffold on hMSC osteogenic differentiation was subsequently examined. SEM images indicate a dense matrix layer deposited on the surface of struts and pore walls. FTIR and XPS measurements show the presence of new functional groups (amide and hydroxyl groups) and elements (C and N) in the ECM/β-TCP scaffold when compared to the β-TCP scaffold alone. Immunofluorescence images indicate that high levels of fibronectin and collagen IV and low level of laminin were present on the scaffold. ECM-containing β-TCP scaffolds significantly increased alkaline phosphatase (ALP) specific activity and up-regulated expression of osteogenesis-related genes such as runx2, alkaline phosphatase, osteopontin and osteocalcin in hMSC, compared to β-TCP scaffolds alone. This increased effect was due to the activation of MAPK/ERK signaling pathway since disruption of this pathway using an ERK inhibitor PD98059 results in down-regulation of these osteogenic genes. Cell-derived ECM-containing calcium phosphate scaffolds is a promising osteogenic-promoting bone void filler in bone tissue regeneration.

Dentin Regeneration Using Deciduous Pulp Stem/Progenitor Cells

Reparative dentin formation is essential for maintaining the integrity of dentin structure during disease or trauma. In this study, we investigated stem/progenitor cell-based tissue engineering for dentin regeneration in a large animal model. Porcine deciduous pulp stem/progenitor cells (PDPSCs) were mixed with a beta-tricalcium phosphate (β-TCP) scaffold for dentin regeneration. Different concentrations of PDPSCs were tested to determine the optimal density for dentin regeneration. Aliquots of 5×105 PDPSCs in 1 mL resulted in the highest number of cells attached to the scaffold and the greatest alkaline phosphatase activity. We labeled PDPSCs with green fluorescent protein (GFP) and used the optimal cell numbers mixed with β-TCP to repair pulp chamber roof defects in the premolars of swine. Four weeks after transplantation, GFP-positive PDPSCs were observed in PDPSC-embedded scaffold constructs. At 16 weeks after transplantation, the PDPSCs mixed with β-TCP significantly regenerated the dentin-like structures and nearly completely restored the pulp chamber roof defects. This study demonstrated that the PDPSC/scaffold construct was useful in direct pulp-capping and provides pre-clinical evidence for stem/progenitor cell-based dentin regeneration.

Improving Bioactivity of Porous β-TCP Ceramics by Forming Bone-Like Apatite Layer on the Surfaces of Pore Walls

Bone tissue engineering provides a new way to repair the bone defect in orthopaedics. The scaffolds, porous materials with excellent biocompatibility, bioactivity and biodegradability, play an important role in bone tissue engineering. Furthermore, the bioactivity of the pore interior surfaces is very important for cell attachment, differentiation and growth, as well as new bone tissue ingrowth into pores. In this paper, β-TCP was selected as materials of scaffolds, and its bioactivity was improved by activating the interior surfaces of pore walls. The porous β-TCP scaffolds with about 50~300μm of pore size and above 80% of porosity were obtained by 3D-gel-laminated processing. Their surfaces of the scaffolds were easily covered by a low crystallized bone-like apatite layer, which determined by XRD and FTIR, after immersing in 1.5SBF solution following pre-treatment by NaOH solution. MTT and ALP assays were performed after cells cultured on the porous scaffolds with bone-like structure, and the results showed higher proliferation rate and differentiation level than that on the scaffolds without treatment, which indicated that the porous β-TCP scaffolds with bone-like apatite layer on surfaces of pore walls possess higher bioactivity. Therefore, the bioactivity of tissue engineering scaffolds could be improved by deposited bone-like apatite layer on their surfaces.

Experimental repair of segmental bone defects in rabbits by angiopoietin‐1 gene transfected MSCs seeded on porous β‐TCP scaffolds

Segmental bone defect repair remains a clinical and experimental challenge in tissue engineering with increasing focus on angiogenesis in the bone substitutes. The objective of this study was to investigate the osteogenic effects of angiopoietin-1 (Ang-1) gene transfected bone marrow-derived mesenchymal stem cells (MSCs) seeded on porous β-TCP scaffolds. This bone substitute (experimental group) and MSCs/β-TCP compounds (control group) were implanted into 15 mm segmental bone defects of the radii of 30 New Zealand white rabbits, with platelet-rich plasma injected at the same time. Bone regeneration and angiogenesis were assessed by Scanning electron microscope (SEM), X-ray, histology, immunohistology, and biomechanical outcome measurements made on the 2nd, 4th, 8th, and 12th week after the operation. In vitro, the amount of proliferation and differentiation of Ang-1 gene transfected MSCs was found to be gross increased than that of the control groups. In vivo, a significantly increased amount of new bone formation accompanied by active capillary vasculature regeneration was observed in the pores of the scaffolds which had been seeded with Ang-1 gene transfected MSCs, as compared with the control groups. The biomechanical test confirmed the failure load of new born bone was close to normal bone. These results suggest that transfer of gene encoding Ang-1 to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation in segmental bone defects.

Design and production of sintered β-tricalcium phosphate 3D scaffolds for bone tissue regeneration

The characteristics of sintered β-tricalcium phosphate (β-TCP) scaffolds produced by 3D printing were studied by means of X-ray diffraction, Scanning Electron Microscopy, Fourier transform infrared spectroscopy, uniaxial compression tests and cytotoxicity tests, using human osteoblast cells.The results reported include details of the β-TCP scaffolds' porosity, density, phase stability, mechanical behavior and cytotoxic profile. Collectively, these properties are fundamental for the future application of these scaffolds as bone substitutes for individualized therapy.

The healing of critical-size calvarial bone defects in rat with rhPDGF-BB, BMSCs, and β-TCP scaffolds

Platelet-derived growth factor-BB (PDGF-BB) plays important roles in regenerating damaged tissue. In this study we investigated the effects of a tissue-engineered bone combined with recombinant human PDGF-BB (rhPDGF-BB), bone marrow stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) to repair critical-size calvarial bone defects in rat. Proliferation and osteogenic differentiation of BMSCs treated with different concentration rhPDGF-BB (0, 10, and 50 ng/ml) was evaluated by MTT, alkaline phosphatase (ALP) activity, alizarin red staining and real-time quantitative PCR (RT-qPCR) analysis of osteogenic gene. BMSCs were then combined with rhPDGF-BB-loaded β-TCP and transplanted into 5 mm calvarial bone defects. The new bone formation and mineralization was evaluated by micro-computerized tomography (Micro-CT) and histological analysis at week 8 after operation. It was observed that the proliferation of BMSCs treated with rhPDGF-BB was enhanced with a time- and dose- dependent manner. There were increased ALP activity, mineralized deposition and elevated mRNA levels of osteogenic gene for BMSCs treated with rhPDGF-BB, particularly in the 50 ng/ml group. Histological analysis showed new bone formation and mineralization in the rhPDGF-BB/BMSCs/β-TCP group was significantly higher than BMSCs/β-TCP, rhPDGF-BB/β-TCP, and β-TCP alone group (P < 0.05). In conclusion, rhPDGF-BB/BMSCs/β-TCP is a promising tissue-engineered bone for craniofacial bone regeneration.

Effects of offset values of solid freeform fabricated PCL–β-TCP scaffolds on mechanical properties and cellular activities in bone tissue regeneration

Scaffolds used in bone tissue regeneration should have highly porous micro-architecture. In addition, they should have high mechanical properties and be biodegradable and biocompatible. In this respect, the solid freeform fabricated PCL–β-TCP scaffold has been widely applied in bone tissue regeneration. In this study, we designed PCL–β-TCP (20 wt%) scaffolds with various offset values, so that the fabricated scaffolds showed five different pore sizes that were dependent on the offset value (0%, 25%, 50%, 75%, and 100%), which showed very similar porosity (about 62%), a square pore shape, and 100% pore interconnectivity. The fabricated scaffolds were assessed not only for physical properties, including calculated bending modulus and water-uptake ability, but also for biological capabilities by culturing osteoblast-like cells (MG63) for various offset values of the scaffolds. Compared with the no-offset scaffolds, the offset scaffolds (especially those with offset values of 50% and 100%) showed superior physical and biological improvements, such as higher bending modulus (maximum increase of 7%), cell-seeding efficiency, cell viability (increase of about 60%), ALP activity (increase of about 50%), and calcium deposition.

3D-printing of highly uniform CaSiO3 ceramic scaffolds: preparation, characterization and in vivo osteogenesis

Calcium silicate (CaSiO3, CS) ceramics have received significant attention for application in bone regeneration due to their excellent in vitro apatite-mineralization ability; however, how to prepare porous CS scaffolds with a controllable pore structure for bone tissue engineering still remains a challenge. Conventional methods could not efficiently control the pore structure and mechanical strength of CS scaffolds, resulting in unstable in vivo osteogenesis. The aim of this study is to set out to solve these problems by applying a modified 3D-printing method to prepare highly uniform CS scaffolds with controllable pore structure and improved mechanical strength. The in vivo osteogenesis of the prepared 3D-printed CS scaffolds was further investigated by implanting them in the femur defects of rats. The results show that the CS scaffolds prepared by the modified 3D-printing method have uniform scaffold morphology. The pore size and pore structure of CS scaffolds can be efficiently adjusted. The compressive strength of 3D-printed CS scaffolds is around 120 times that of conventional polyurethane templated CS scaffolds. 3D-Printed CS scaffolds possess excellent apatite-mineralization ability in simulated body fluids. Micro-CT analysis has shown that 3D-printed CS scaffolds play an important role in assisting the regeneration of bone defects in vivo. The healing level of bone defects implanted by 3D-printed CS scaffolds is obviously higher than that of 3D-printed β-tricalcium phosphate (β-TCP) scaffolds at both 4 and 8 weeks. Hematoxylin and eosin (H&E) staining shows that 3D-printed CS scaffolds induce higher quality of the newly formed bone than 3D-printed β-TCP scaffolds. Immunohistochemical analyses have further shown that stronger expression of human type I collagen (COL1) and alkaline phosphate (ALP) in the bone matrix occurs in the 3D-printed CS scaffolds than in the 3D-printed β-TCP scaffolds. Considering these important advantages, such as controllable structure architecture, significant improvement in mechanical strength, excellent in vivo osteogenesis and since there is no need for second-time sintering, it is indicated that the prepared 3D-printed CS scaffolds are a promising material for application in bone regeneration.

The Osteogenic Study of Tissue Engineering Bone with BMP2 and BMP7 Gene-Modified Rat Adipose-Derived Stem Cell

To evaluate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat adipose-derived stem cells (ADSCs), modified with BMP2 and BMP7 by lentivirus. In the present study, ADSCs transfected with Lv-BMP2 and Lv-BMP7, alone or together, were seeded on β-TCP scaffold and cultured in vitro. Based on the results of DNA assay, alkaline phosphatase (ALP) activity, alizarin red staining and osteogenic marker genes expression analysis, the BMP2 and BMP7 genes cotransfection group exhibited a higher degree of osteogenic differentiation in vitro. To investigate the in vivo osteogenesis of the tissue engineering bone, the ADSCs/β-TCP constructs were implanted in rat femurs defects for 6 weeks and studied histomorphology and radiography. The results showed that BMP2 and BMP7 genes cotransfection group dramatically enhanced the efficiency of new bone formation than BMP2 group and BMP7 group in vivo. These results demonstrated that it was advantageous to construct tissue engineering bone using ADSCs cotransfected with BMP2 and BMP7 on β-TCP, providing a potential way for treating bone defects.

Fabrication and properties of porous β-tricalcium phosphate ceramics prepared using a double slip-casting method using slips with different viscosities

A new method to enhance the flexural strength of porous β-tricalcium phosphate (β-TCP) scaffolds was developed. This new method provides better control over the microstructures of the scaffolds and enhances the scaffolds’ mechanical properties. Using this technique, we were able to produce scaffolds with mechanical and structural properties that cannot be attained by either the polymer sponge or slip-casting methods alone or by simply combining the polymer sponge and slip-casting methods. The prepared scaffolds had an open, uniform, interconnected porous structure with a bimodal pore size of 100.0–300.0μm. The flexural strength of the bimodal porous β-TCP scaffold sintered at 1200°C was 56.2MPa and had porosity of 61.4vol%. The scaffolds obtained provide good mechanical support while maintaining bioactivity, and hence, these bioscaffolds hold promise for applications in hard-tissue engineering.

Co-culturing mesenchymal stem cells from bone marrow and periosteum enhances osteogenesis and neovascularization of tissue-engineered bone

Mesenchymal stem cells (MSCs) isolated from bone marrow and periosteum are often used as cellular sources for bone tissue engineering. This study showed that co-cultured human bone marrow stem cells (hBMSCs) and periosteal-derived stem cells (hPCs) resulted in a synergistic effect on osteogenic differentiation both in vitro and in vivo. Compared to hBMSCs and hPCs, co-culturing MSCs showed abundant mineralization, robust calcium deposition, steadily increasing ALP activity, and upgraded mRNA expression of osteogenic specific genes (COL1A1, BMP-2, osteopontin, osteocalcin) in vitro. Eight weeks after implantation of cellular β-TCP scaffolds in immunodeficient mice, similar synergistic effects were confirmed during in vivo evaluation of total new bone formation, mature bone formation, and neovascularization. Based on these findings, the use of co-cultured hBMSCs and hPCs can be recommended as a promising new approach for bone tissue engineering applications.

Freeze Drying Technology Forming Drug Release Bionic Scaffolds with Gradient Structure

A kind of drug release bionic scaffold with gradient structure was designed to release drugs. The scaffold mould had been designed with CAD software and fabricated with rapid prototype. The bionic beta-tricalcium phosphate (β-TCP) scaffold was fabricated with freeze drying. It was composed of two coaxial cylindrical porous scaffolds. The internal loose scaffold was embedded in the outer dense layer. The two different density layers of the scaffold could be used to adjust the drug releasing rate through regulating the slurry concentration. The scaffold was distributed with pores ranged from 30μm to 100μm. The research indicated that the scaffold with gradient structure could be loaded with two kinds of drugs which were released at different rate. The scaffolds were fit as the substitute materials for bone repairing.

Low-dose corticosteroid and regenerative cell therapies for acute laminitis.

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 β-tricalcium phosphate (β-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/β-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 β-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.

Adipose-derived stem cells transfected by adenoviral vector mediated human transforming growth factor-β1 gene with β-tricalcium phosphate scaffold in vitro

Objective To observe the biological behaviors of cultured adipose-derived stem cells (ADSCs) transfected by adenoviral vector-mediated human transforming growth factor-β1 (Ad-hTGF-β1) combined with β-tricalcium phosphate (β-TCP) scaffold and investigate the feasibility of them for cartilage tissue engineering. Methods The adenoviral plasmid vector containing hTGF-β1 gene was transfected into ADSCs. The ADSCs were seeded onto the β-TCP scaffold and the effects of adhesion and morphological changes were observed under the phase-contrast microscopy and scanning electron microscopy (SEM). Reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were applied to detect the expression of collagen Ⅱ in the β-TCP scaffold. Results The SEM results showed the porous β-TCP scaffold had macro- and micro-porous strutures with the porosity being 65%. The ADSCs transfected by Ad-hTGF-β1 were successfully cultured in vitro. The induced cells adhered to the surface of the β-TCP scaffold and proliferated well. RT-PCR, Western blotting and immunohistochemistry revealed that Collagen Ⅱ was detected after mixed culture. Conclusion The porous β-TCP scaffold with excellent property may be a good matrix for ADSCs transfected by Ad-hTGF-β1, and can be used for cartilage engineering. Key words: Adipose-derived stem cell; β-tricalcium phosphate; Scaffold; Tissue engineering

Improvement of mechanical properties of macroporous β-tricalcium phosphate bioceramic scaffolds with uniform and interconnected pore structures

The biocompatible and degradable macroporous bioceramic scaffolds with high mechanical properties and interconnected porous structures play an important role in hard tissue regeneration and bone tissue engineering applications. In this study, the improvement of mechanical properties of macroporous β-tricalcium phosphate [β-Ca 3(PO 4) 2, β-TCP] bioceramic scaffolds with uniform macropore size and interconnected pores were fabricated by impregnation of the synthesized β-TCP nano-powder slurry into polymeric frames. The microstructures, mechanical properties and in vitro degradation of the fabricated samples were investigated. For a comparison, β-TCP scaffolds were also fabricated from commercial micro-size powders under the same conditions. The resultant scaffolds showed porosities ∼65% with uniform macropore size ranging from 400 to 550 μm and interconnected pore size ∼100 μm. The compressive strength of the samples fabricated from nano-size powders reached 10.87 MPa, which was almost twice as high as those fabricated from commercial micro-size powders, and was comparable to the high-end value (2–10 MPa) of human cancellous bone. Furthermore, the degradation of the β-TCP bioceramics fabricated from nano-size powders was apparently lower than those fabricated from commercial micro-size powders, suggesting the possible control of the degradation of the scaffolds by regulating initial powder size. Regarding the excellent mechanical properties and porous structures, the obtained macroporous β-TCP bioceramic scaffolds can be used in hard tissue regeneration and bone tissue engineering applications.

Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading.

The role of perfusion bioreactor in proliferation and distribution of bone marrow stromal cells in a large-scale scaffold

Objective To study the role of perfusion bioreactor in proliferation and distribution of rat bone marrow stromal cells (BMSCs) in a large-scale scaffold. Methods SD rat BMSCs transfected with enhanced green fluorescent protein (eGFP) (eGFP-BMSCs) were planted in large-scale porous β-tricalcium phosphate (β-TCP) scaffolds. In the dynamic perfusion culture group, the scaffold with eGFP-BMSCs was continuously cultured in our self-designed three-dimensional perfusion bioreactor for 7, 14 and 28 days. In the static culture group, the scaffold was put into a medium reservoir without perfusion for 7, 14 and 28 days.Proliferation and distribution of the cells in the scaffold were examined by scanning electronic microscopy (SEM), fluorescence microscopy (FM), measuring daily glucose consumption, and counting eGFP-BMSCs in each layer. Results SEM and FM showed that eGFP-BMSCs distributed and proliferated throughout the scaffold in dynamic perfusion culture, but distributed and proliferated only in the peripheral pores of the scaffold in static culture. The daily glucose consumption in both groups increased with time. Cell proliferation reached the plateau phase after culture for 14 days in the static culture group, but after culture for 21 days in the perfusion culture group. The rate and margin of increase were much more evident in the perfusion culture group. On day 28, glucose consumption in the perfusion culture group was 36. 33 ± 3. 14 mg/d, 3. 7 times as large as that in the static culture group (9. 82 ± 1. 33 mg/d). The eGFP-BMSCs counting revealed there were no significant differences in cell number between layers of the scaffold on days 7 and 28 d ( P > 0. 05), but there were significant differences on day 14 in the perfusion culture group ( P < 0. 05); while in the static culture group, there were significant differences in cell number between layers of the scaffold ( P < 0. 05),with most of the cells assembled at the bottom of the scaffold.Conclusion Our self-designed three-dimensional perfusion bioreactor may help eGFP-BMSCs proliferate and distribute uniformly in a large-scale porous scaffold. Key words: Bone marrow cells; Bioreactor; Cell culture techniques; Tissue engineering

Enhanced mechanical performance and biological evaluation of a PLGA coated β-TCP composite scaffold for load-bearing applications

Porous β-tricalcium phosphate (β-TCP) has been used for bone repair and replacement in clinics due to its excellent biocompatibility, osteoconductivity, and biodegradability. However, the application of β-TCP has been limited by its brittleness. Here, we demonstrated that an interconnected porous β-TCP scaffold infiltrated with a thin layer of poly(lactic-co-glycolic acid) (PLGA) polymer showed improved mechanical performance compared to an uncoated β-TCP scaffold while retaining its excellent interconnectivity and biocompatibility. The infiltration of PLGA significantly increased the compressive strength of β-TCP scaffolds from 2.90 to 4.19 MPa, bending strength from 1.46 to 2.41 MPa, and toughness from 0.17 to 1.44 MPa, while retaining an interconnected porous structure with a porosity of 80.65%. These remarkable improvements in the mechanical properties of PLGA-coated β-TCP scaffolds are due to the combination of the systematic coating of struts, interpenetrating structural characteristics, and crack bridging. The in vitro biological evaluation demonstrated that rat bone marrow stromal cells (rBMSCs) adhered well, proliferated, and expressed alkaline phosphatase (ALP) activity on both the PLGA-coated β-TCP and the β-TCP. These results suggest a new strategy for fabricating interconnected macroporous scaffolds with significantly enhanced mechanical strength for potential load-bearing bone tissue regeneration.

The optimum flow shear stress and the mass transport benefiting for construction of the tissue-engineered bone combining computational fluid dynamics

Objective To explore the optimum flow shear stress and mass transport for the construction of tissue-engineered bone.Methods The β-tricalcium phosphate (β-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (HBMMSCs) were cultured in perfusion bioreactor.When the same flow rate was applied,the flow shear stress was separately 1×,2× and 3×.When the same flow shear stress was applied,the flow rates were separately 3 ml/min,6 ml/min and 9 ml/min.Cell proliferation was measured by MTT method.The construction of tissue-engineered bone was evaluated by measuring alkaline phosphatase (AKP) activity,secretion of osteopontin (OP) and osteocalcin (OC),and the mineralization of extracellular matrix (ECM).The flow shear stress and the mass transport were obtained using computational fluid dynamics.Results When the flow rate was same,the most cell proliferation was found in 2× group.The AKP activity and secretion of OC was higher in 2× and 3× groups than in those in 1× group.After 28days,the highest amount of mineralization of ECM was found in 3× group.When the flow shear stress was same,the AKP activity was highest in 6 ml/min group.After 28 days,secretion of OC and formation of mineralized ECM was highest in 3 ml/min group.When the flow rate was same,the flow shear stress was separately 0.004-0.007 Pa,0.009-0.013 Pa and 0.013-0.018 Pa.When the flow shear stress was same,the flow rate was separately 0.267-0.384 mm/s,0.521-0.765 mm/s and 0.765-1.177 mm/s.Conclusion When the tissue-engineered bone was constructed,0.013-0.018 Pa flow shear stress and 0.267-0.384 mm/s mass transport velocity could improve the construction of the tissue-engineered bone in vitro. Key words: Tissue engineering; Mesenchymal stem cells; Bioreactors; Biological transport

In Vivo Efficacy of Bone Marrow Stromal Cells Coated with Beta-Tricalcium Phosphate for the Reconstruction of Orbital Defects in Canines

To repair the segmental orbital rim defects of dogs with three-dimensional (3D) tissue-engineered constructs derived from culturing autogenous bone marrow stromal cells (BMSCs) on β-tricalcium phosphate (β-TCP) scaffolds. A 25-mm segmental defect on the canine inferior orbital rim was created. BMSCs were isolated and osteogenically induced in vitro, then were seeded onto 3D β-TCP scaffolds and implanted to repair the orbital defects after 5 days of cultivation. The group of noninduced BMSC/β-TCP, β-TCP alone, and the normal inferior orbital rim were set as controls. The orbits of all groups had spiral computed tomography (CT) scans 1, 4, 8, and 12 weeks after surgery. Gross examination, bone density, microCT, and histologic measurements were performed 12 weeks after surgery. The results were analyzed to evaluate the extent of bone repair. Twelve weeks after surgery, CT examination revealed good inferior orbital rim recovery in the induced BMSC/β-TCP group, and the bone density was 0.30 ± 0.03 g/cm(2) with no dominant variance, compared with the normal control (P > 0.05). MicroCT and histologic examination confirmed that the implantations led to good repair of the defects. Pore-like spongy bone surrounded the implants through the section plane, with some residue remaining in the center. In contrast, the noninduced BMSC/β-TCP implants were not fully repaired, and nonunion was evident. The bony density for this group was 0.23 ± 0.07 g/cm(2), which was significantly lower than that of the control group (P < 0.05). The β-TCP group was largely held by fibrous tissues. Engineered bone from induced BMSCs and 3D biodegradable β-TCP can efficiently repair critical-sized segmental orbital defects in dogs.