Shao Zengwu

Molecular tissue engineering: Concepts, status and challenge

Tissue engineering has confronted many difficulties mainly as follows: 1) How to modulate the adherence, proliferation, and oriented differentiation of seed cells, especially that of stemcells. 2) Massive preparation and sustained controllable delivery of tissue inducing factors or plasmid DNA, such as growth factors, angiogenesis stimulators, and so on. 3) Development of “intelligent biomimetic materials” as extracellular matrix with a good superficial and structural compatibility as well as biological activity to stimulate predictable, controllable and desirable responses under defined conditions. Molecular biology is currently one of the most exciting fields of research across life sciences, and the advances in it also bring a bright future for tissue engineering to overcome these difficulties. In recent years, tissue engineering benefits a lot from molecular biology. Only a comprehensive understanding of the involved ingredients of tissue engineering (cells tissue inducing factors, genes, biomaterials) and the subtle relationships between them at molecular level can lead to a successful manipulation of reparative processes and a better biological substitute. Molecular tissue engineering, the offspring of the tissue engineering and molecular biology, has gained an increasing importance in recent years. It offers the promise of not simply replacing tissue, but improving the restoration. The studies presented in this article put forward this new concept for the first time and provide an insight into the basic principles, status and challenges of this emerging technology.

Protective effect of niacinamide on interleukin-1β-induced annulus fibrosus type II collagen degeneration in vitro

The protective effect of niacinamide on interleukin-1β (IL-1β)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba’s intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1β) and treatment group (niacinamide+IL-1β). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type II collagen degeneration group (P<0.01). Type II collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type II collagen in treatment group was significantly stronger than that in type II collagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1β stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1β-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

Surface modification of biomimetic PLGA-(ASP-PEG) matrix with RGD-containing peptide: a new non-viral vector for gene transfer and tissue engineering

RGD-containing peptide (K16-GRGDSPC), characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP-PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were used to detect the MW and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%–95%, and MS shows that the MW was 2 741.3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur (C/S) was 99.746:0.1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/S was 99.574:0.4255, and there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

Up-regulation of niacinamide in intervertebral disc aggrecanin vitro

SummaryThe regulatory effects of niacinamide (Nia) on intervertebral disc (IVD) aggrecanin vitro was investigated. Chibas 10 ng/mL interleukin-1 (IL-1)-induced rabbit IVD degeneration modelin vitro was established. 0. 5, 0. 25 and 0. 05 mg/mL Nia was added to normal and degenerated IVDs for intervention. On the first and second week after intervention, safranin O-fast green staining intensity and glycosaminoglycan (GS) content were measured. The expression of aggrecan core protein was detected by RT-PCR. The results showed: (1) After treatment with 0.5 mg/mL Nia for one week, the GS content in nucleus pulposus (NP) was increased by 44.8%, as compared with control group (P<0.01); The GS content in IL-1 induction groups was increased with the increase of Nia concentrations: After treatment with 0.5 mg/mL for one week, the GS content in NP was increased by 68.3 % as compared with control group (P<0.01). After two weeks, GS content in NP and fibrous rings was still higher than in control group at the same period (P<0.01) and untreated group (P<0.01). (2) Safranin O-fast green staining revealed that with the increase of Nia concentrations staining density in NP and fibrous rings was increased and histological structure damage to IVDs by IL-1β was alleviated. (3) RT-PCR showed that the expression of core protein gene in IL-1β-induced degenerated IVDS was increased with the increase of Nia concentrations. It was concluded that under conditionsin vitro, Nia could up-regulate the expression of aggrecan in IVDs and protect IVDs from IL-1β-induced degeneration at least partially, which offers a potential choice for IVD degeneration clinical therapy.

Treatment of floating knee injury in children.

SummaryThe necessity and superiority of the surgical operation on children with floating knee injury and the fracture union and complications were investigated. Twenty-eight children with floating knee injury were subjected to open reduction and internal fixation or external fixator. The patients were followed up for 18 months to 7 years. The curative effectiveness was scored by Karlstrom criteria. The results showed that no nonunion or deformity was found. The affected limb was 1.2 cm to 1.5 cm longer in 2 cases, 0.8 to 1.2 cm shorter in 3 cases than the contralateral. No severe dysfunction of knee joint occurred. The excellent-good rate was 92.8% and the curative rate 71.4% respectively. So for children whose age is older than 5 years, its a good way to treat the fractures of femur and tibia with open reduction and internal fixation or external fixator. The method can be advantageous for the nursing care, early function recovery, shortening of the hospital stay and avoidance of severe complications.

Effect of exogenous p16ink4a and hRb1 genes on cell cycle regulation of osteosarcoma cell

SummaryTo study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRbl, PIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectivelyin vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P<0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alonein vitro.

Altersbezogene Haufigkeitsquoten und Ermittlung der Osteophyten des Wirbelkorpers

ZusammenfassungObjective Eine Datenbank der Normwerte der Hufigkeitsquoten der Osteophyten an den Lendenwirbelkörpern aufzubauen. Methoden 633 Röntgen-Übersichtsaufnahmen von männlichen Personen im Alter zwischen 20 und 87 Jahren und 607 Röntgen-Übersichtsaufnahmen von weiblichen Personen im Alter zwischen 20 und 92 Jahren ausgewertet. Eegebnisse Die Häufigkeitsquoten der Osteophyten nahmen mit zunehmendem Lebensalter zu. In den Altersgruppen 50–59 Jahren und 60–69 Jahren lagen die Häufigkeitsquoten der Osteophyten bei Männern signifikant höher als bei Frauen (P<0.01;P<0.05). Bei Männern waren am Wirbelkörper L4 am häufigsten Osteophyten vorhanden, gefolgt vom Wirbelkörper L3; bei Frauen waren am Wirbelkörper L3 am häufigsten Osteophyten vorhanden, gefolgt vom Wirbelkörper L4. Folgerung Mit Hilfe der Datenbasis ist es möglich, die Häufigkeitsquoten der Osteophyten an den Lendenwirbelkörpern durch Vergleich mit der altersbezogenen Norm quantitativ zu bewerten.

Construction of antisense transforming growth factorβ1 gene and its effect on the proliferation by expression in osteosarcoma cells

SummaryTo construct the antisense transforming growth factorβ1 (TGFβ1) gene and investigate the effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGFβ1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGFβ1(−). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.