ABSTRACT
This study aimed to analyze the expression of bone matrix proteins and CD31 by immunohistochemistry after maxillary sinus grafting with different bioimplants in a rabbit model. Rabbit demineralized bone matrix (DBM), partially purified bovine bone morphogenetic proteins (BMP), a mixture of BMP with DBM (BMP/DBM), or particulated autogenous bone was grafted into the maxillary sinuses of 42 rabbits. Animals were sacrificed at 2 and 8 weeks. Immunohistochemistry was used to investigate the expression of type 1 collagen (COL1), osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN), and CD31. Sinuses grafted with BMP were filled with trabeculae of woven bone that was strongly immunoreactive for COL1, OC, ON, and BSP. BMP/DBM showed strongly positive immunoreactivity for these proteins within the newly formed bone, but weak immunoreactivity in the DBM particles. Immunoreactivity for COL1, OC, ON, and BSP in DBM sinuses was only seen in the osteoblasts rimming the grafted bone particles. The staining of autogenous bone graft sinuses was similar to those grafted with DBM. OPN staining was detected in autogenous bone graft, BMP/DBM, and BMP bioimplants. CD31 staining was strongest in BMP and BMP/noncollagenous matrix proteins sinuses. These results suggest that exogenous BMP enhances not only osteogenesis but also angiogenesis, an important part of bone repair.
Subject(s)
Biocompatible Materials/pharmacology , Bone Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Implants, Experimental , Maxillary Sinus/metabolism , Maxillary Sinus/pathology , Models, Animal , Animals , Bone Matrix/drug effects , Bone Matrix/pathology , Cattle , Collagen Type I/metabolism , Immunohistochemistry , Integrin-Binding Sialoprotein/metabolism , Male , Maxillary Sinus/drug effects , Osteocalcin/metabolism , RabbitsABSTRACT
This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.
Subject(s)
Bone Marrow Cells/cytology , Bone Matrix/metabolism , Bone Morphogenetic Protein 4/pharmacology , Bone Morphogenetic Proteins/pharmacology , Cell Differentiation , Platelet-Rich Plasma/metabolism , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Proteins/metabolism , Cattle , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Humans , Integrin-Binding Sialoprotein , Neovascularization, Physiologic/drug effects , Osteogenesis/drug effects , Osteopontin/genetics , Osteopontin/metabolism , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Stromal Cells/cytology , Stromal Cells/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
OBJECTIVE: To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. METHODS: Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. RESULTS: The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the alkaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblasts in vivo and in differentiating osteoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. CONCLUSION: Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type I collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.
Subject(s)
Bone Matrix/metabolism , Bone Morphogenetic Proteins/pharmacology , Gene Expression Regulation/physiology , Osteoblasts/drug effects , Osteoblasts/metabolism , Alkaline Phosphatase/metabolism , Animals , Cattle , Cell Line , DNA/metabolism , MiceABSTRACT
OBJECTIVE: To investigate the potentially useful of platelet-rich plasma (PRP) on mRNA expression of angiogenesis. STUDY DESIGN: Adjunct assay and reverse-transcription polymerase chain reaction (RT-PCR) analysis of type I collagen, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) in rat bone marrow stromal cells differentiation in 14 days' culture. RESULTS: The PRP significantly elevated alkaline phosphatase activity after day 5 (P < .05), and DNA and protein content increased at culture days 1, 3, and 5 (P < .01) with PRP compared with control. The RT-PCR demonstrated that type I collagen was expressed in all substrates and remained high with PRP during 14 days of culture, and that mRNA expression of VEGF and PDGF were higher over time. CONCLUSIONS: This study indicates a potential contribution of PRP as possibly starting the process of angiogenesis, recruiting the endothelial cells which line blood vessels, and beginning the initiation of bone regeneration.
Subject(s)
Bone Marrow Cells/metabolism , Platelet-Derived Growth Factor/biosynthesis , Platelet-Rich Plasma , Vascular Endothelial Growth Factor A/biosynthesis , Animals , Cell Differentiation , Cells, Cultured , Collagen Type I/biosynthesis , Gene Expression , Male , Neovascularization, Physiologic , RNA, Messenger/biosynthesis , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/metabolismABSTRACT
Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of certain BMPs to promote bone graft and fracture healing. To observe the mechanism of osteoinductive and bone formation, 100 microg of bovine BMP was tested during osteogenic differentiation of rat bone marrow stromal cells (MSCs) and C2C12 line culture for 14 and 28 days. We examined alkaline phosphatase (ALP) by assay, immunohistochemical studies for bone matrix proteins, and mRNA expression of bone matrix proteins and osteoblast-related analysis by reverse-transcription polymerase chain reaction. ALP activity in MSC cultures was elevated by bovine BMP by two to fivefold (P < 0.05-0.001). DNA and protein content increased over 14 days. BMP significantly increased the mRNA expression of type I collagen, ALP, osterix, osteocalcin, osteopontin, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-A, and parathyroid hormone receptor time dependently during the osteoblastic differentiation. There was no markedly enhanced mRNA expression of bone sialoprotein (BSP) and glyceraldehyde-3-phosphate dehydrogenase compared with that of control. Immunohistochemical results also showed BMP increased immunoreactive positivity of type I collagen, osteocalcin, osteonectin, osteopontin, and BSP during the C2C12 differentiation. These data indicated that BMP enhances our ability to stimulate the differentiation of osteoblast-like cells and increases osteoinductivity, bone matrix protein formation and mineralization, angiogenesis, and chondrogenesis during osteoblast progenitor cell differentiation in vitro and that the role of chondrogenic is weak.
