Kazuma Fujimura

Correlation between pain and synovitis in patients with internal derangement of the temporomandibular joint

A correlation between pain scores by questionnaire and synovitis index via arthroscopic inspection was made in 28 patients with internal derangement of the temporomandibular joint (TMJ) with closed lock. A statistically significant but weak correlation was found between the visual analog scale of pain and the index of distribution of synovitis. However, in the questionnaire, pain on chewing had a positive correlation with both the intensity and distribution of synovitis in the TMJ. This study indicates that synovitis is one cause of TMJ pain in patients with closed lock.

Osteoinduction by recombinant human bone morphogenetic protein-2 at intramuscular, intermuscular, subcutaneous and intrafatty sites

To compare the osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) at various sites in rats, 5 microg of rhBMP-2 were implanted into various sites, using atelopeptide type-I collagen (CL) as a carrier (BMP groups). CL implantation was used as a control. Forty Wistar rats were divided into intramuscular, intermuscular, subcutaneous and intrafatty site groups (IrM, IeM, SC and IF, respectively). Bone formation was evaluated radiographically, histologically and biochemically 21 days after implantation. In the BMP groups, the alkaline phosphatase activity and calcium content at all sites were higher than those in the control groups. Among the BMP groups, the new bone formation was highest in the IrM and lowest in the IF radiographically, histologically and biochemically. Five microg of rhBMP-2, a relatively low dose, induced adequate new bone formation in all sites. The variations of osteoinductive activity of rhBMP-2 in various sites may be due to differences in the blood supply.

Osteoinduction capability of recombinant human bone morphogenetic protein-2 in intramuscular and subcutaneous sites: an experimental study

The osteoinduction capability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in the muscle and in the subcutaneous tissue in Wistar rats (n = 20) was evaluated, using atelopeptide type-I collagen as a carrier. The alkaline phosphatase (ALP) activity and calcium (Ca) content were quantitatively analyzed 1, 3, 7 and 21 days after the implantation of 5 micrograms of rhBMP-2. At 3 days, the ALP activity began to increase gradually. The Ca content showed a slow increase until 7 days and was markedly elevated at 21 days. There was no significant difference observed between the intramuscular and subcutaneous sites until 3 days. However, at 7 days, both the ALP activity and Ca content were significantly higher intramuscularly than subcutaneously. Also, at 21 days they were higher in the muscle than in the subcutaneous tissue. These results suggest that the difference in osteoinduction could be related to the partial pressure of oxygen or the blood supply in the intramuscular and subcutaneous sites, and that immature mesenchymal cells in the muscle could more easily differentiate into osteoblasts, leading to osteoinduction. This study clearly demonstrated that even a small amount (5 micrograms) or rhBMP-2 induces new bone in the subcutaneous tissue, which has a lesser blood flow than the muscle.

Enhancement by Recombinant Human Bone Morphogenetic Protein-2 of Bone Formation by Means of Porous Hydroxyapatite in Mandibular Bone Defects

Hydroxyapatite is osteoconductive and can maintain an original biocompatible form. It is useful, in the reconstruction of bone defects, to enhance the osteoconduction of hydroxyapatite with an osteogenic protein. The aim of this study was to evaluate the bone formation in surgically created defects of rabbit mandibles by a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2), with porous hydroxyapatite and atelopeptide type I collagen used as the carrier for rhBMP-2. A 10-μg rhBMP-2-implanted group (n = 15) and a control group (n = 15), in which only atelopeptide type I collagen and porous hydroxyapatite were implanted, were histologically examined 3, 7, and 21 days after implantation. The alkaline phosphatase activity was also quantitatively analyzed. No new bone formation was observed in either the tested or the control group after 3 days. At 7 days, immature bone tissue was observed in some pores of the rhBMP-2implanted group, while in the control group, immature mesenchymal cells were observed. At 21 days, trabecular bone lined some pore walls. In the central portion, the bone marrow, including angioid tissue, was observed. New trabecular bone formation was observed on portions of the external surface of the hydroxyapatite disk. On the other hand, the control group showed infiltration of immature mesenchymal cells into some pores. Marginal bone formation was found in the pores close to the surface of the disk which opposed mandibular bone. The control group showed a slow, small increase in alkaline phosphatase activity in this study, while the experimental group showed a marked increase at 21 days. This increase was significantly higher in the tested group than in the control group at both 7 and 21 days. The findings indicate that rhBMP-2 accelerated bone formation by osteoconduction from porous hydroxyapatite. The combination of rhBMP-2, atelopeptide type I collagen, and porous hydroxyapatite is suggested to be advantageous for clinical application in reconstructing mandibular bone defects.

In Vitro and in Vivo Studies of a Bone Morphogenetic Protein-2 Expressing Adenoviral Vector

Background: Bone morphogenetic proteins (BMPs) play important roles in the migration of osteoblast progenitor cells, the proliferation of mesenchymal cells, and their differentiation into chondrogenic and osteogenic cells. However, the optimum procedure to deliver BMPs remains unknown. To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo. Methods: C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector (AxCALacZ). Twenty-four hours after the infection, indirect immunofluorescence was performed. On day 5 after the infection, alkaline phosphatase (ALP) in the cells and osteocalcin in the culture medium were measured. Furthermore, to examine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated osteoinduction by AxCAOBMP-2, under transient immunosuppression with cyclophosphamide, given at a dose of 125 mg/kg intraperitoneally the day before injection of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2 (8.75 108 plaque-forming units [pfu], Group I) and AxCALacZ (1.75 108 pfu, control group) and 5 l of AxCAOBMP-2 (1.75 108 pfu, Group II) were injected into a right calf muscle of Wistar rats. On day 21, bone formation in each group was investigated radiologically and histologically. Results: Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cells transferred with the BMP-2 gene by this vector produced ALP in the cells and also produced and secreted osteocalcin in the culture medium. Osteoinduction was found only in the AxCAOBMP-2 treated groups with immunosuppression. Osteoinduction activity was higher in Group I than in Group II. Conclusion: This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene. Clinical Relevance: Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.

Self-regenerating bone implant: ectopic osteoinduction following intramuscular implantation of a combination of rhBMP-2, atelopeptide Type I collagen and porous hydroxyapatite

A combination of recombinant human bone morphogenetic protein-2 (rhBMP-2), atelopeptide Type I collagen (CL) as a carrier and porous hydroxyapatite (pHAP) was implanted in a calf muscle pouch of the rat. Three rhBMP-2-implanted groups (2, 10 and 50 micrograms; each n = 5) and the control group (n = 5), in which only CL and pHAP were implanted, were established. Three weeks later, the implants were examined.

Osteogenesis by recombinant human bone morphogenetic protein-2 at skeletal sites.

Osteogenesis was evaluated in the mandibular bone by combinations of various dosages of recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite (four groups: Group I, 2 μg recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group II, 10 μg recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Group III, 50 μg recombinant human bone morphogenetic protein-2, atelopeptide Type I collagen, and porous hydroxyapatite; Control Group, only atelopeptide Type I collagen and porous hydroxyapatite). The prepared materials were implanted in the mandibular bone hole (7 mm in diameter, 2 mm deep). Three weeks later, the alkaline phosphatase activity in the implanted region was determined, and the histologic features of the excised tissue were examined. There were significant differences in histologic and biochemical findings among the four groups. In the recombinant human bone morphogenetic protein-2 implanted groups, osteogenesis increased with the dosage of recombinant human bone morphogenetic protein-2, as assessed by alkaline phosphatase activity and histologic findings. The results suggest that atelopeptide Type I collagen is an effective carrier for recombinant human bone morphogenetic protein-2 and that porous hydroxyapatite would be advantageous for clinical application as a material to maintain its original form after implantation.

Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp

Populations of pluripotent stem cells were isolated from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous teeth and their multipotentiality properties compared. Osteogenic, chondrogenic, adipogenic, and neurogenic differentiation potentials were examined. Bone marrow mesenchymal stem cells (BMMSCs) and synovial fluid-derived cells (SFCs) showed the highest levels of osteogenesis as expressed by alkaline phosphatase (ALP) activity (0.54±0.094 U/mg protein and 0.57±0.039 U/mg protein, respectively; P=0.60) and by osteocalcin (BGLAP; determined by real-time RT-PCR). SFCs showed the highest levels of chondrogenesis as expressed by ALP activity (1.75±0.097 U/mg protein) and of COL2A1 and COL10A1 by real-time PCR. In terms of adipogenesis, lipid vesicles were observed in the BMMSCs and SFCs. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) exhibited neurogenesis potential, as shown by increases in expression of class III β-tubulin (TUBB3) and microtubule-associated protein 2 (MAP2) on RT-PCR. Variability was found in the differentiation potential corresponding to the tendency of the original tissue to differentiate. It is suggested that the cell type should be selected depending on the regenerative treatment regimen.

Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder

AIMS Mesenchymal stem cells are an interesting source of material for regenerative medicine. The present study aimed at characterizing the phenotype and differentiation potential of adherent synovial fluid-derived cells from temporomandibular joint (TMJ) disorder patients. MAIN METHODS Synovial fluid collection takes place during TMJ cavity irrigation arthrocentesis under local anesthesia. The synovial fluid-derived adherent cells were fibroblast-like and spindle-shaped. Ex vivo-expanded synovial fluid-derived cells were shown to express STRO-1 and CD146, previously found to be present in bone marrow mesenchymal stem cells. Further, they were identified as being capable of differentiating into a variety of cell types including osteoblasts, chondrocytes, adipocytes, and neurons. KEY FINDINGS The present study demonstrates that human pluripotent cells can be isolated from synovial fluid. These synovial fluid-derived cells cannot only be derived from a very accessible resource, but are also capable of providing sufficient cells for potential clinical applications. SIGNIFICANCE These cells may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

The effect of blood supply in muscle and an elevated muscle flap on endogenous tissue-engineered bone by rhBMP-2 in the rat.

&NA; Metabolism and remodeling of bony tissue are maintained and controlled by blood supply. In this study, bony tissue osteoinduced heterotopically by recombinant human bone morphogenetic protein‐2 (rhBMP‐2) in rat muscles with different amounts of blood supply was investigated. The implant beds were in the latissimus dorsi muscle (LDM) and in the space wrapped with an elevated latissimus dorsi muscle flap (LDMF). The blood flow was preestimated at two muscle sites using a laser Doppler blood flowmeter. The flow value of the LDM was 2.23 ± 0.17 ml/min/100 g (mean ± standard deviation). The flow values of the LDMF were 1.71 ± 0.22 ml/min/100 g 30 minutes after elevating the LDMF, 1.75 ± 0.20 ml/min/100 g after 1 week, 1.83 ± 0.19 ml/min/100 g after 2 weeks, and 1.99 ± 0.18 ml/min/100 g after 3 weeks. Bony tissues induced heterotopically by rhBMP‐2 were examined radiographically and histologically. On radiographs, radiopaque shadows in the LDM were almost as large as those in the LDMF. The radiopacity in the LDM was a little higher than that of the LDMF. The microscopic findings showed increased trabeculae and more hematopoietic marrow in the LDM than lamellalike bone in the LDMF. Mean bony area in the implant was 1.05 ± 0.26 mm2 in the LDM, and was 0.70 ± 0.11 mm2 in the LDMF. Bony proportions in the overall implant area were 18.3 ± 3.46% in the LDF and 12.1 ± 2.18% in the LDMF. The current study indicates that blood supply is an important factor for promoting heterotopic osteoinduction by rhBMP‐2 to produce massive bony tissue as an endogenous method of tissue engineering. Kusumoto K, Bessho K, Fujimura K, Akioka J, Okubo Y, Ogawa Y, lizuka T. The effect of blood supply in muscle and an elevated muscle flap on endogenous tissue‐engineered bone by rhBMP‐2 in the rat. Ann Plast Surg 2000;45:408‐414