The objective of this program is to investigate the biological effect of dynamic strain on human periosteal cells in vitro. Using a well-established model, the Flexercell unit, we placed mechanical stress (50,000 microstrain, 1 Hz and sine wave) on human periosteal cells grown in collagen coated flexible membrane. The time points of proliferative and differentiative properties were assessed by means of cell counting, thymidine incorporation, synthesis of alkaline phosphatase and osteocalcin, and long term of mechanical load induced calcium nodules formation was also demonstrated. The results showed that the application of highly controlled strains exerted a significant effect on human periosteal cells by up regulation of osteogenic properties rather than exercised an influence on proliferation. The results suggested that the promoting effects of dynamic strain on human periosteal cells probably contribute to the biological function of mechanical loading bearing.
Biomechanical Phenomena ; Cell Proliferation ; Cells, Cultured ; Humans ; Periosteum ; cytology ; Stress, Mechanical ; Ulna ; cytology ; Weight-Bearing

BACKGROUNDRepair of large bone defects remains a challenge for clinicians. The present study investigated the ability of mesenchymal stem cells (MSCs) and/or periosteum-loaded poly (lactic-co-glycolic acid) (PLGA) to promote new bone formation within rabbit ulnar segmental bone defects.

METHODSRabbit bone marrow-derived MSCs (passage 3) were seeded onto porous PLGA scaffolds. Forty segmental bone defects, each 15 mm in length, were created in the rabbit ulna, from which periosteum was obtained. Bone defects were treated with either PLGA alone (group A), PLGA + MSCs (group B), periosteum-wrapped PLGA (group C) or periosteum-wrapped PLGA/MSCs (group D). At 6 and 12 weeks post-surgery, samples were detected by gross observation, radiological examination (X-ray and micro-CT) and histological analyses.

RESULTSGroup D, comprising both periosteum and MSCs, showed better bone quality, higher X-ray scores and a greater amount of bone volume compared with the other three groups at each time point (P < 0.05). No significant differences in radiological scores and amount of bone volume were found between groups B and C (P > 0.05), both of which were significantly higher than group A (P < 0.05).

CONCLUSIONSImplanted MSCs combined with periosteum have a synergistic effect on segmental bone regeneration and that periosteum plays a critical role in the process. Fabrication of angiogenic and osteogenic cellular constructs or tissue-engineered periosteum will have broad applications in bone tissue engineering.

Animals ; Bone Regeneration ; physiology ; Cells, Cultured ; Lactic Acid ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; cytology ; Polyglycolic Acid ; chemistry ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

Patient own fibrin may act as the safest, cheapest and immediate available biodegradable scaffold material in clinical 1 tissue engineering. This study investigated the feasibility of using patient own fibrin isolated from whole blood to construct a new human cartilage, skin and bone. Constructed in vitro tissues were implanted on the dorsal part of the nude mice for in vivo maturation. After 8 weeks of implantation, the engineered tissues were removed for histological analysis. Our results demonstrated autologous fibrin has great potential as clinical scaffold material to construct various human tissues.
*Biocompatible Materials ; *Bone Transplantation ; Cartilage/*transplantation ; Cell Division/physiology ; Culture Media ; *Fibrin ; Fibroblasts/cytology ; Mesenchymal Stem Cells/cytology ; Mice, Nude ; Organ Culture Techniques ; Periosteum/cytology ; *Skin Transplantation ; *Tissue Engineering

The strategy used to generate tissue-engineered bone construct, in view of future clinical application is presented here. Osteoprogenitor cells from periosteum of consenting scoliosis patients were isolated. Growth factors viz TGF-B2, bFGF and IGF-1 were used in concert to increase cell proliferation during in vitro cell expansion. Porous tricalcium phosphate (TCP)-hydroxyapatite (HA) scaffold was used as the scaffold to form 3D bone construct. We found that the addition of growth factors, greatly increased cell growth by 2 to 7 fold. TCP/HA proved to be the ideal scaffold for cell attachment and proliferation. Hence, this model will be further carried out on animal trial.
Bone Regeneration/*physiology ; *Bone Transplantation ; Cell Division/physiology ; Collagen/metabolism ; *Mesenchymal Stem Cell Transplantation ; Organ Culture Techniques ; Periosteum/*cytology ; Tissue Engineering/*methods

OBJECTIVETo evaluate the therapeutic effect of bone morphogenetic protein 2 (BMP-2) gene modified tissue engineering bone (GMB) combined with vascularized periosteum in the reconstruction of segmental bone defect.

METHODSAdenovirus carrying BMP-2 gene (Ad-BMP-2) was transfected into the isolated and cultured rabbit bone marrow stromal cells (MSCs). The transfected MSCs were seeded on bovine cancellous bone scaffolds (BCB) to construct gene modified tissue engineering bone (GMB). The bilateral rabbits radial defects (2.5 cm long) were created as animal model. The rabbits were divided into five groups to reconstruct the defects with CMB combined with vascularized periosteum (group A); or GMB combined with vascular bundle implantation (group B); or GMB combined with free periosteum (group C); or GMB only (group D); or BCB scaffolds only (group E). Angiogenesis and osteogenesis were observed by X-ray, histological examination, biomechanical analysis and capillary ink infusion.

RESULTSIn group A, the grafted GMB was revascularized rapidly. The defect was completely reconstructed at 8 weeks. The mechanism included both intramemerbrane and endochondral ossification. In group B, the vascular bundle generated new blood vessels into the grafted GMB, but the osteogenesis process was slow in the central zone, which healed completely at 12 weeks. In group C, the free graft of periosteum took at 4 weeks with angiogenesis. The thin extremal callus was formed at 8 weeks and the repairing process almost finished at 12 weeks. Better osteogenesis was found in group D than in group E, due to the present of BMP2 gene-transfected MSCs. The defects in group D were partial repaired at 12 weeks with remaining central malunion zone. The defects in group E should nonunion at 12 weeks with only fibre tissue.

CONCLUSIONSBMP-2 gene modified tissue engineering bone combined with vascularized periosteum which provides periosteum osteoblasts as well as blood supply, has favorable ability of osteogenesis, osteoinduction and osteoconduction. It is an ideal method for the treatment of segmental bone defect.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Bone and Bones ; pathology ; Cattle ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; blood supply ; transplantation ; Rabbits ; Surgical Flaps ; blood supply ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transfection

OBJECTIVETo investigate the practicability of periosteum construction in vitro by small intestinal submucosa (SIS) as a tissue scaffold with bone marrow mesenchymal stem cells (BMSC).

METHODSThe bone marrow mesenchymal stem cells were cultivated by the conventional method in vitro, and then co-cultivated with SIS. The morphological feature of the complex material, the extracellular matrices, the adhesion and proliferation of BMSC were observed by optical microscope, electronic microscope, scanning electronic microscope and histologic evaluation respectively.

RESULTSBMSCs adhered and proliferated on SIS, secreted a great deal of extracellular matrices with active cellular function. BMSCs grew well there in layers, and the thickness became thicker as time passed, acting like a biological periosteum.

CONCLUSIONCombined culture of SIS and BMSC in vitro can construct a tissue engineering periosteum similar to the natural one, thus providing a base for further study of its osteogenic regeneration in vivo.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Intestinal Mucosa ; anatomy & histology ; Intestine, Small ; anatomy & histology ; Membranes, Artificial ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; Rabbits ; Swine ; Tissue Engineering ; methods

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.
Biocompatible Materials ; Bone Development ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Collagen ; chemistry ; Humans ; Lactic Acid ; chemistry ; Microscopy, Fluorescence ; Models, Statistical ; Osteogenesis ; Periosteum ; metabolism ; Polyglycolic Acid ; chemistry ; Polymers ; chemistry ; Stem Cells ; cytology ; Tissue Engineering

OBJECTIVETo observe the effectiveness of prevention and cure for maxillary growth deformity following tissue engineered oral mucosa implantation on mucoperiosteal denuded palate process in young rat.

METHODSHard palate mucoperiosteum of a SD baby rat were excised and oral keratinocytes were isolated and cultured. Tissue engineered oral mucosa was fabricated with the cultured oral keratinocytes and the membrane made of sodium alginate (SA). 80 female three-week-old SD rats were used as subjects in this study. The animals were divided randomly into a normal control group and 3 experimental groups, each group included 20 rats. Normal control group (NG) were not operated. Hard palate mucoperiosteum on left side in all experimental groups were excised, exposed bone were not treated in denuded group (DG), but repaired with membrane in material group (MG) and repaired with the tissue engineered oral mucosa in mucosal group (MUG). All the animals were sacrificed at 9th week postoperatively (12 weeks old), and the clean widths of right and left hard palatal were measured under a dissection microscope. The difference between palatal widths of two sides and the asymmetry ratio between the different groups were compared and analyzed.

RESULTSNo significant difference in asymmetry was discovered between the DG and the MG, but the asymmetry in MUG was less than DG or MG.

CONCLUSIONTissue engineered oral mucosal implantation in palatoplasty is an effective method in preventing and curing secondary maxilla deformity by repairing denuded bone wound.

Animals ; Animals, Newborn ; Cleft Palate ; physiopathology ; surgery ; Keratinocytes ; cytology ; Maxilla ; growth & development ; Mouth Mucosa ; transplantation ; Oral Surgical Procedures ; methods ; Palate, Hard ; surgery ; Periosteum ; surgery ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering