e State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of Agricultural Ministry, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
c State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100190, China
E-mail: [email protected]
d Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu 213164, China
a Department of Orthopedics, Zhengzhou Orthopedics Hospital, Zhengzhou, Henan, China
f Department of Oncology, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
E-mail: [email protected]
b Department of Spine Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116011, China
E-mail: [email protected]
To advance the “bottom-up” approaches for bone tissue engineering, we developed rat bone mesenchymal stem cell (BMSC)-gelatin microspheres (BGMs) as cell-culture modules and collagen binding domain-bone morphogenetic protein2 (CBD-BMP2)-collagen microcarriers (CCMs) as differentiation-control modules. BMSCs were efficiently seeded onto gelatin microspheres in a spinner flask to form BGMs with a high cell density. CBD-BMP2 was recombined and bound with collagen microfibers to form CCMs, in which BMP2 was controllably released. BGM and CCM were assembled as building blocks to fabricate macroscopic bone-like constructs in vitro and in vivo, where BMSCs were directly induced to differentiate into osteocytes. The osteogenic differentiation of BMSCs was confirmed with increasing mineral deposition (alkaline phosphatase stain, alizarin red stain, calcium content and micro-CT), gene expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and collagen type I) as well as alkaline phosphatase activity. The in vivo formed bone-like tissues had the compression modulus of 2.62 ± 1.41 MPa, similar to that in the in vivo bone tissues, much higher than those in BGM samples of 0.03 ± 0.012 MPa. Our results suggesting these proof-of-concept strategies, assembling cell-culture modules and function-control modules to modularly engineer clinical-relevant large tissue replacements, have great potential applications in tissue engineering.
Videos of PTZ-induced seizures were scored offline with a standard seizure severity scale appropriate for generalized seizures (Pohl and Mares, 1987) using Observer Video-Pro software (Noldus, Wageningen, The Netherlands). The seizure scoring scale was divided into stages as follows: 0, no change in behavior; 0.5, abnormal behavior (sniffing, excessive washing, and orientation); 1, isolated myoclonic jerks; 2, atypical clonic seizure; 3, fully developed bilateral forelimb clonus; 3.5, forelimb clonus with tonic component and body twist; 4, tonic-clonic seizure with suppressed tonic phase with loss of righting reflex; and 5, fully developed tonic-clonic seizure with loss of righting reflex (Pohl and Mares, 1987).
Cortical tissue was dissected from the brains of adult (postnatal day >21) male and female Wistar Kyoto rats and stored separately at −80°C until use. Tissue was suspended in a membrane buffer, containing 50 mM Tris-HCl, 5 mM MgCl2, 2 mM EDTA, and 0.5 mg/ml fatty acid-free bovine serum albumin (BSA) and complete protease inhibitor (Roche, Mannheim, Germany), pH 7.4, and was then homogenized using an Ultra-Turrax blender (Labo Moderne, Paris, France). Homogenates were centrifuged at 1000g at 4°C for 10 min, and supernatants were decanted and retained. Resulting pellets were rehomogenized and centrifugation was repeated as before. Supernatants were combined and then centrifuged at 39,00g at 4°C for 30 min in a high-speed Sorvall centrifuge; remaining pellets were resuspended in membrane buffer, and protein content was determined by the method of Lowry et al. (1951). All procedures were carried out on ice.
To investigate neuronal excitability in vitro, we used both the Mg 2+ -free and 4-AP models of epileptiform activity in acute hippocampal brain slices, as measured using MEA electrophysiology. Two separate models of epileptiform activity were used to provide a broader analysis of drug effects (Hill et al., 2009; Whalley et al., 2009). Hippocampal slices have a well defined architecture ( Fig. 1 A), exhibited no spontaneous LFP events in control aCSF, and proved readily amenable to MEA recording ( Fig. 1 B). We sought to take advantage of the ability of MEAs to record spatiotemporal activity at multiple discrete, identifiable regions by investigating activity at CA1, CA3, and DG regions within the hippocampus. Application of Mg 2+ -free aCSF ( Fig. 1 C) or 4-AP aCSF ( Fig. 1 D) to hippocampal slices resulted in the appearance of robust spontaneous epileptiform LFPs across the preparation. LFPs were consistent with status epilepticus-like activity and were reliably recorded using the multisite MEA technique ( Table 1 ). Slice-to-slice variability and electrode contact variability resulted in substantial variation in signal strength ( Table 1 ); therefore, subsequent drug-induced changes in burst characteristics were normalized to control bursts before drug application (these analyses are fully characterized in Hill et al., 2009).
Receptor Binding Assays
Substrate-integrated MEAs (Multi Channel Systems, Reutlingen, Germany) (Egert et al., 2002a; Stett et al., 2003) were used to record spontaneous neuronal activity as described previously (Ma et al., 2008). MEAs were composed of 60 electrodes (including reference ground) of 30 μm diameter, arranged in an ∼8 × 8 array with 200 μm spacing between electrodes.