Yasuharu Yamazaki

Response of the Mouse Femoral Muscle to an Implant of a Composite of Bone Morphogenetic Protein and Plaster of Paris

A composite consisting of 1 mg of bone morphogenetic protein (BMP) and 5 mg of plaster of Paris (PLP) was implanted into the mouse femoral muscle. Control PLP without BMP was implanted into the contralateral muscle. The BMP/PLP composite induced cartilage formation within two weeks, trabecular bone within three weeks, and lamellar bone including bone marrow within six weeks. PLP did not disturb BMP-induced bone formation. In addition, the BMP/PLP composite was resorbed early after implantation. These results suggest that PLP composite system is one of the most suitable BMP delivery systems currently available.

Bone morphogenetic protein encapsulated with a biodegradable and biocompatible polymer.

To develop a controlled release system for bone morphogenetic protein (BMP), poly(DL-lactide-co-glycolide) (PLGA) capsules containing BMP were prepared by an interfacial precipitation method using a water-in-oil-in-water emulsion. The surface morphology, particle size distribution, and hydrolytic degradation rate of the PLGA capsules were examined. The encapsulation yield and release rate of BMP in vitro were measured using fluorescein isothiocyanate-labeled BMP. The amount of BMP released from PLGA capsules increased between days 3 and 5. In addition, the effectiveness of BMP encapsulated in PLGA to induce bone formation in vivo was also examined by subcutaneous implantation in rats. Complete digestion of the capsules and new bone formation including bone marrow were identified by histologic examination of harvested tissues at 3 weeks after implantation. These results demonstrated that encapsulation of BMP with PLGA could be a promising method to induce bone in clinics.

The role of recombinant human bone morphogenetic protein-2 in PLGA capsules at an extraskeletal site of the rat

Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor-beta (TGF-beta) superfamily and has strong bone-inductive activity in vivo. To examine the role of BMP-2 in an extraskeletal site of rat using a controlled release system of peptides, we encapsulated the recombinant human BMP-2 (rhBMP-2) with poly(DL-lactide-co-glycolide) (PLGA) and implanted the rhBMP-2/PLGA capsules in the subcutaneous area of rats. Upon histochemical examination, it was found that bone-inducing cells having alkaline phosphatase (ALP) activity appeared around the capsules by the suitably released rhBMP-2. In addition, the temporal histological examination showed that direct bone formation without cartilage occurred in the process of this ectopic bone induction. These data indicate that the role of rhBMP-2 in the extraskeletal site of rats is to induce the differentiation of mesenchymal cells into the osteoblasts.

Restoration of segmental bone defects in rabbit radius by biodegradable capsules containing recombinant human bone morphogenetic protein-2.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLGA) capsules to regenerate bone by controlling the release rate of rhBMP-2. The rhBMP-2/PLGA capsules containing 12 microg of rhBMP-2 were implanted in seven 15-mm segmental defects of rabbits radii to examine the healing capacity of the rhBMP-2/PLGA capsules. For the control group, four segmental defects were left empty and two were implanted with ghost PLGA capsules. Healing of the defects was followed for 24 weeks and periodically evaluated by radiographs and histological examination. Mechanical testing was applied to three regenerated bone samples at 24 weeks postoperatively when the mature cortex was observed. Mechanical properties of regenerated bone were not significantly different from normal intact bone statistically. Histologically, the rhBMP-2/PLGA capsules disappeared completely during the process of bone regeneration. These results increased possibilities for clinical application of rhBMP-2/PLGA capsules.

Bone regeneration produced in rat femur defects by polymer capsules containing recombinant human bone morphogenetic protein-2

PURPOSE This study examines the effect of polymer capsules containing recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone regeneration in a large segmental bone defect in the rat. MATERIALS AND METHODS Poly(DL-lactide-co-glycolide) (PLGA) capsules containing 3 microg of rhBMP-2 were implanted in a 5-mm segmental femoral defect in rats, and the femurs were harvested at 4 and 8 weeks after implantation and observed by radiographically and microscopically. RESULTS At 8 weeks after implantation, union of bone was found radiographically and histologically in the femoral defects implanted with the rhBMP-2/PLGA capsules. In contrast, the control animals did not show bridging of the defect. CONCLUSIONS This study provides evidence that use of rhBMP-2/PLGA capsules is a promising delivery system to regenerate bone.

Ectopic induction of cartilage and bone by bovine bone morphogenetic protein using a biodegradable polymeric reservoir

Two kinds of bone morphogenetic protein (BMP) containing polymeric reservoirs made from poly(lactide-co-glycolide) (PLG) and a mixture of PLG/poly(ethylene glycol) (PEG) (PLG/PEG) were implanted into mice femoral muscles. In the PLG/PEG reservoir with BMP, bone formation was induced within 3 weeks. On the other hand, in the PLG reservoir with BMP scarcely any bone formation could be observed. The results suggest that the carrier system is most important for the development of BMP-induced bone formation and the PLG/PEG reservoir system is available for BMP delivery.

Osteogenic potential of cryopreserved human bone marrow-derived mesenchymal stem cells cultured with autologous serum.

Secondary bone grafting in the alveolar cleft is one of the most important therapeutic modalities for patients with cleft lip and palate. However, in children, harvesting a sufficient amount of bone is difficult, and repeated operations are often required because deformation of the alveolar cleft may occur because of the grafted bone absorption and bone growth, which imposes a heavy burden on the patients. The burden may be reduced if the banking of human bone marrow-derived mesenchymal stem cells (MSCs) could be made possible, that is, if cryopreserved autologous MSCs, those that have been harvested from the patients own bone marrow, could be cultured and expanded with the patients own serum and can be thawed and cultivated for grafting at a later date. In the current study, a hybrid-type bone substitute was prepared by thawing and cultivating MSCs that have been cryopreserved for more than 3 months. The hybrid-type bone substitute was implanted subcutaneously in nude mice. At 6 and 9 weeks after grafting, the bone graft was removed, and the osteogenic potential of the cells cultured with autologous serum, as determined by alkaline phosphatase activity and alizarin red S staining, was compared with those cultured with fetal bovine serum. There was no significant difference in the osteogenic potential between MSCs cultured with autologous serum and those cultured with fetal bovine serum. The results suggest the possibility of artificial bone grafting using MSCs cultured with autologous serum and the banking of the cells.

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: A preliminary study

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB.

Sequence analysis of fibroblast growth factor receptor 2 ( FGFR2 ) in Japanese patients with craniosynostosis.

Recently, mutations of the fibroblast growth factor receptor (FGFR) genes have been detected in syndromic craniosynostosis. We examined nucleotide sequences of FGFR2 in Japanese craniosynostosis patients (Crouzon syndrome: 9 cases; Apert syndrome: 6 cases; scaphocephaly: 3 cases as non-syndromic patients) by polymerase chain reaction (PCR) followed by direct sequencing methods. The results demonstrated FGFR2 heterozygous mutations at codons 252, 290 of exon 7, and at codon 342, 354 of exon 9 in Crouzon syndromes. In Apert syndrome patients, Ser252Trp and Pro253Arg were detected in five and one patients, respectively. No mutation was detected in one case of Crouzon, all cases of scaphocephaly and healthy individuals. Thus far sequence analysis of FGFR2 in syndromic craniosynostosis has been reported in many white patients, whereas in Japanese only several cases have been studied. The current study with 18 patients confirmed that a similar series of mutations occur in Japanese patients as in white patients regardless of ethnicity and environment. The frequency of the mutation was 82% (9/11 cases) in Japanese Crouzon patients. The ratio of S252W:P253R was 5 : 1 in Japanese Apert patients. Morever, in Japanese Apert patients, complication rate of cleft palate was 60% for mutation of Ser252Trp and 0 of 2 patients for Pro253Arg, with their syndactyly score being 4.90 and 5.50, respectively.

The effect of preoperative use of an orthopedic plate on articulatory function in children with cleft lip and palate

Objective To evaluate the effect of preoperative use of an orthopedic plate (OP) on postoperative articulatory function in children with cleft lip and palate. Subjects The subjects had complete unilateral or bilateral cleft lip and palate and were scheduled for a one-stage palatoplasty. Main Outcome Measures Tongue movements during sucking were analyzed by ultrasonography. Postoperative articulatory behavior was also assessed at 5 years 4 months of age. Results There was an excessive downward excursion of the rear portion of the tongue during sucking regardless of the use or nonuse of the OP. This indicated that infants with cleft palate could not create negative pressure in the oral cavity, even with the OP. However, the OP appeared effective for preventing irregular movements of the tongue during sucking. The proportion of subjects obtaining excellent articulation was significantly higher in the group using the OP until palatoplasty than in the group who did not continue using the OP. The proportion of subjects with disturbed articulatory function in the latter group was comparable with that in the control group, who never used the OP. Conclusions Continuous use of the OP up to the time of palatoplasty appeared to be effective for the postoperative articulatory function in children with complete cleft lip and palate. Inhibiting irregular movements of the tongue, the OP might assist in preventing “palatalized articulation.”