The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rabbits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as control. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts, trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the surface of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good biocompatibility and ectopic induced osteogenic activity.
Animals ; Biocompatible Materials ; Bone Marrow Cells ; cytology ; Bone Matrix ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Decalcification Technique ; Osteogenesis ; Rabbits ; Stem Cells ; cytology ; Stromal Cells ; cytology ; Tissue Engineering

The biocompatibility and osteogenic activity of allogenic decalcified bone matrix (DBM) used as a carrier for bone tissue engineering were studied. Following the method described by Urist, allogenic DBM was made. In vitro, DBM and bone marrow stromal cell (BMSC) from rabbits were co-cultured for 3-7 days and subjected to HE staining, and a series of histomorphological observations were performed under phase-contrast microscopy and scanning electron microscopy (SEM). In vivo the mixture of DBM/BMSC co-cultured for 3 days was planted into one side of muscules sacrospinalis of rabbits, and the DBM without BMSC was planted into other side as control. Specimens were collected at postoperative week 1, 2 and 4, and subjected to HE staining, and observed under SEM. The results showed during culture in vitro, the BMSCs adherent to the wall of DBM grew, proliferated and had secretive activity. The in vivo experiment revealed that BMSCs and undifferentiated mesenchymal cells in the perivascular region invaded gradually and proliferated together in DBM/BMSC group, and colony-forming units of chondrocytes were found. Osteoblasts, trabecular bone and medullary cavity appeared. The inflammatory reaction around muscles almost disappeared at the second weeks. In pure DBM group, the similar changes appeared from the surface of the DBM to center, and the volume of total regenerate bones was less than the DBM/BMSC group at the same time. The results indicated that the mixture of DBM and BMSC had good biocompatibility and ectopic induced osteogenic activity.
Biocompatible Materials ; Bone Marrow Cells/*cytology ; Bone Matrix/*cytology ; Cells, Cultured ; Chondrocytes/cytology ; Coculture Techniques ; Decalcification Technique ; *Osteogenesis ; Stem Cells/cytology ; Stromal Cells/cytology ; *Tissue Engineering

OBJECTIVETo investigate the feasibility of using marrow stromal osteoblast-cancellous bone matrix compound artificial bone (MCCAB) as tissue-engineered bone, the osteogenesis of MCCAB in the cranial defect was observed in the experiment.

METHODSThe in vitro cultivated and induced marrow stromal cells of adult New Zealand rabbits were seeded into the alginate-cancellous bone matrix to form MCCAB. The MCCAB was then implanted into the cranial defect for 4 to 8 weeks. The cancellous bone matrix (CBM) alone or the marrow stromal osteoblasts (MSOs) alone was implanted as the control. The effectiveness of bone formation was assessed by histological and roentgenographic analysis.

RESULTSThe osteogenesis of MCCAB was better than CBM or MSOs and superior to the blank group.

CONCLUSIONMCCAB can effectively repair cranial defect. It could be used clinically to restore large bone defects.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Bone Matrix ; cytology ; Cells, Cultured ; Feasibility Studies ; Male ; Osteoblasts ; cytology ; physiology ; Rabbits ; Skull ; abnormalities ; Stromal Cells ; cytology ; physiology

OBJECTIVETo evaluate application of the sponge of demineralized bone matrix (SDBM) in tissue engineering of bone.

METHODSSDBM was prepared from long bone of rabbits. Bone marrow cells were flushed from the bone shaft of femurs of a two-month-old New Zealand white rabbit. After the cells were cultured for 9 days, the flasks were added into dexamethasone (10(-8) mol/L), beta-glycerophosphate sodium (10 mmol/L) and L-ascorbic acid (50 micrograms/ml). After 5 weeks, the cultured cells were collected and marked by 5-Bromo-2'-dexyouridine (BrdU). The grand sum of cells seeded on a piece of SDBM was about (4-6) x 10(6). The composites of cells and SDBM (tissue engineered chip, TEC) were implanted into muscles and bone defects of radius in rabbits. A standard procedure was applied to make a 10 mm long defect bilaterally in the radius of nine skeletally mature male New Zealand white rabbits. All of the 18 defects were randomly divided into three groups: group I, six defects were grafted by TEC; group II, six defects were grafted with SDBM alone; group III, six defects were empty.

RESULTSThe results of radiographic and histological evaluation showed that all of the defects were repaired in group I and group II at 6 weeks, none of the defects was repaired in group III. The results of BrdU staining showed that the staining was positive in group I, but negative in group II. Biomechanical test showed that the compressive ultimate strength (CUS) of new bone in TEC implanted group was comparable with normal radius (P = 0.623) and in SDBM implanted group was significant lower than normal radius (P = 0.038).

CONCLUSIONSThe TEC can form cartilage and bone tissue in muscles and repair segmental bone defects. SDBM is a kind of effective natural scaffold in tissue engineering of bone.

Animals ; Bone Demineralization Technique ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Bone Matrix ; Implants, Experimental ; Male ; Rabbits ; Radius Fractures ; surgery ; Random Allocation ; Stem Cells ; cytology ; Tissue Engineering

Matrix metalloproteinases (MMPs) are endocellular proteolytic enzymes. They are so named because they need Ca2+, Zn2+ and other metal ions as their cofactors. MMPs play an important biological role in regulating the formation, remodeling and degradation of extracellular matrix and participate in various physiological and pathological processes of cells. Bone marrow-derived mesenchymal stem cells (BMSCs) are a kind of pluripotent stem cell which has the ability to self-renew and differentiate into functional cells. Meanwhile, they can respond to the damage signals and migrate to injured site for tissue repair and regeneration. MMPs and their inhibitors TIMPs affect the differentiation and migration of BMSCs. This article reviews the roles of MMPs in differentiation and migration of BMSCs.
Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Humans ; Matrix Metalloproteinases ; physiology ; Mesenchymal Stromal Cells ; cytology

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is essential to the attachment, proliferation and differentiation of bone cells (osteoblast, osteoclast and osteocytes) and the formation of bone tissue. However, conventional scaffold materials for bone tissue engineering proved less valuable for actual applications because they lack mechanical strength, interconnected channel network, and controllable porosity or channel size. Therefore,to explore the ideal scaffold materials is one of the popular studies on current bone tissue engineering. In this paper, we review, the application and advancement of a newly-developed technology generally known as rapid prototyping (RP) techniques in bone tissue engineering.
Bone Substitutes ; Bone and Bones ; Cell Differentiation ; Cell Division ; Cells, Cultured ; Extracellular Matrix ; Humans ; Osteoblasts ; cytology ; Porosity ; Tissue Engineering

OBJECTIVETo observe the osteoinductive activity of demineralized bone matrix (DBM) and deproteinized bone (DPB) made from human avascular necrotic femoral head.

METHODSThe femoral head was cut into pieces with the size of 3 mm x 3 mm x 5 mm, which were made into DBM and DPB. These two kinds of biomaterials were cocultured with human bone mesenchymal stem cells (hBMSCs). Monolayer cells without biomaterials were cultured as control. Proliferative activity of hBMSCs was evaluated on days 1, 3, 5, 7 and 14. The concentration of alkaline phosphatase (ALP), osteocalcin (OC), and Ca(2+) were detected on days 1, 7, 14 and 21.

RESULTSCells cultured in DBM showed higher proliferative activity than did in DPB and monolayer cells (F =39.773, P <0.01). DBM and DPB also had osteoinductive activity. The concentrations of ALP (F=93.162, P <0.01), OC (F =236.852, P < 0.01), Ca(2+)(F =80.711, P <0.01) of DBM group were significantly higher than that of DPB and control groups.

CONCLUSIONSIn vitro, DBM and DPB made from avascular necrotic femoral head have osteoinductive activity when cocultured with hBMSCs, and the former is stronger than the latter.

Alkaline Phosphatase ; analysis ; Biocompatible Materials ; Bone Demineralization Technique ; Bone Matrix ; cytology ; Bone and Bones ; cytology ; Calcium ; analysis ; Cell Proliferation ; Cells, Cultured ; Femur Head Necrosis ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteocalcin ; analysis ; Osteogenesis

This paper was designed to investigate the effect of laminar shear stress on matrix metalloproteinase -9 (MMP-9) expression in rat bone marrow-derived mesenchymal stem cells (MSCs), and the possible signal transduction mechanism involved. Rat bone marrow MSCs were isolated and cultured, then, exposed to laminar shear stress at indicated strengths such as low (5dyne/cm2), medium (15 dyne/cm2) and high (30 dyne/cm2) via parallel plate flow chamber. RT-PCR was used to analyze the expression of MMP-9. The signaling inhibitors such as Wortmannin (PI3K specific inhabitor), SB202190 (p38MAPK specific inhabitor), and PD98059 (ERK1/2 specific inhabitor) were used to investigate the possible mechanical signal transduction pathway. The results showed: (1) The expression of MMP-9 was weak in static state, however, MMP-9 expression increased when MSCs were exposed to 15 dyne/cm2 shear stress for 2 hours, and MMP-9 expression increased with the extension of stimulating time, and it reached the peak at 24 h; (2) MSCs were stimulated by shear stress for 2 hours at different strengths (5 dyne/cm2, 15 dyne/cm2, 30 dyne/cm2), and under all these conditions, the expression of MMP-9 increased, and reached the peak at 15 dyne/cm2; (3) After MSCs were pretreated by three kinds of signal pathway inhibitors, the expression of MMP-9 did not change obviously in Wortmannin group and PD98059 group, but it was significantly inhibited in SB202190 group. This study demonstrated that shear stress could induce the expression of MMP-9 in rat bone marrow-derived mesenchymal stem cells; the amount of MMP-9 expression was closely related to stimulating time and the strengths of shear stress; and p38MAPK signal pathway played a critical role during the process.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Signal Transduction ; Stress, Mechanical

To prepare poly(lactic acid/glycolic acid/ asparagic acid-co- polyethylene glycol) (PLGA-[ASP-PEG]) and examine the cellular biocompatibility. PLGA-[ASP-PEG] was obtained by bulk ring-opening copolymerization method, examined by infrared spectrometry (IR) and 1H nuclear magnetic resonance spectroscopy (1H NMR). Bone marrow stromal cells(BMSCs) were cultured with PLGA-[ASP-PEG] (experiment gruop) and PLGA (control group) in vitro respectively, and were observed by phase-contrast microscopy and scanning electron microscopy. The resuls showed that PLGA-[ASP-PEG] was obtained and proved by IR and 1H NMR. The BMSCs of the experiment group could well attach to and extend on the surface of the PLGA-[ASP-PEG], and could proliferate and secrete better extracellular matrix, compared with control. The PLGA-[ASP-PEG] has good cellular a biocompatibility. It can be used as a biomaterial for bone tissue engineering.
Aspartic Acid ; chemistry ; Biocompatible Materials ; Bone Matrix ; cytology ; Lactic Acid ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Polyesters ; Polyethylene Glycols ; chemistry ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Tissue Engineering

OBJECTIVETo evaluate the use of cancellous bone matrix gelatin (BMG) combined with chondrocytes in constructing tissue-engineered cartilage by observing the growth, proliferation and differentiation of chondrocytes on allogeneic cancellous BMG.

METHODSThe articular chondrocytes isolated from a 1-month-old rabbit were multiplied to a monolayer and seeded onto cancellous BMG to construct tissue-engineered cartilage in vitro during a period of 6 weeks. Samples were taken from the construct after 1, 2, 4, and 6 weeks of culture and evaluated by histology, immunohistochemistry and transmission electron microscopy (TEM).

RESULTSThe chondrocytes excreted matrix proteoglycan and collagen on cancellous BMG. With the prolongation of the culture time, the cells proliferated in the construct and the cells in the lacunae increased. Numerous chondrocytes were present the central region of the cancellous BMG and surrounded by extracellular matrix. By 6 weeks of culture, the BMG was covered with 15-20 layers of chondrocytes and cartilaginous tissue occurred in the pores throughout the cancellous BMG. Immunohistochemical staining showed rich and evenly distributed type II collagen around the chondrocytes, and TEM revealed an ultrastructure of the chondrocyte similar to that of native chondroctyes, with abundant extracellular matrix produced around the cells.

CONCLUSIONTissue-engineered cartilage can be constructed in vitro using allogeneic cancellous BMG combined with chondrocytes. Allogeneic cancellous BMG serves as a good scaffold material for tissue-engineered cartilage to promote the growth and proliferation of the seeded chondrocytes and allows maintenance of the differentiation phenotype of the cells.

Absorbable Implants ; Animals ; Bone Matrix ; chemistry ; Cartilage ; cytology ; growth & development ; Cells, Cultured ; Chondrocytes ; cytology ; physiology ; Gelatin ; chemistry ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds