Enhanced Bone Regeneration in Variable-Type Biphasic Ceramic Phosphate Scaffolds Using rhBMP-2.

Our aim was to investigate the bone regeneration capacity of powder-type biphasic ceramic scaffold (BCP powder), block-type BCP (BCP block), and collagen-added block-type BCP (BCP collagen) with different concentrations of recombinant human bone morphogenetic protein 2 (rhBMP-2) in an animal model. Four rabbits were assigned to each of the following groups: no graft + rhBMP-2 (0.1/0.2 mg/mL), BCP powder + rhBMP-2 (0.1/0.2 mg/mL), BCP block + rhBMP-2 (0.1/0.2 mg/mL), and BCP collagen + rhBMP-2 (0.1/0.2 mg/mL), i.e., a total of 32 rabbits. Polycarbonate tubes (Φ 7 mm × 5 mm) for supporting scaffolds were fixed into a 7 mm round border. Subsequently, 0.1 mL of rhBMP-2 solutions with different concentrations was injected into the tubes. Both radiological and histomorphometric analyses showed that osteogenesis was not enhanced by increasing the concentration of rhBMP-2 in all groups at both 3 and 6 weeks. Radiological analysis showed that bone formation was higher in the BCP collagen group than in the BCP powder and BCP block groups at both rhBMP-2 concentrations at 3 weeks. rhBMP-2 enhanced bone formation; however, as the concentration increased, bone formation could not be enhanced infinitely. Collagen-added alloplastic graft material may be useful for mediating rapid bone formation in initial stages.

Cranial Suture Mesenchymal Stem Cells: Insights and Advances.

The cranial bones constitute the protective structures of the skull, which surround and protect the brain. Due to the limited repair capacity, the reconstruction and regeneration of skull defects are considered as an unmet clinical need and challenge. Previously, it has been proposed that the periosteum and dura mater provide reparative progenitors for cranial bones homeostasis and injury repair. In addition, it has also been speculated that the cranial mesenchymal stem cells reside in the perivascular niche of the diploe, namely, the soft spongy cancellous bone between the interior and exterior layers of cortical bone of the skull, which resembles the skeletal stem cells' distribution pattern of the long bone within the bone marrow. Not until recent years have several studies unraveled and validated that the major mesenchymal stem cell population of the cranial region is primarily located within the suture mesenchyme of the skull, and hence, they are termed suture mesenchymal stem cells (SuSCs). Here, we summarized the characteristics of SuSCs, this newly discovered stem cell population of cranial bones, including the temporospatial distribution pattern, self-renewal, and multipotent properties, contribution to injury repair, as well as the signaling pathways and molecular mechanisms associated with the regulation of SuSCs.

Dynamic changes of facial skeletal fractures with time.

To investigate the characteristics of imaging changes with time of facial fractures, patients with facial fractures who had computed tomographic scan were enrolled including 500 patients who were divided into six groups based on the time of scanning: super early (<3 d), early (4-7 d), early-to-medium (8-14 d), medium (15-21d), medium-to-late (22d-2 months) and late stage (>2 months). The data were compared and analyzed. Forty two patients with frontal bone fractures had high-energy impact as the reason of fractures. The fracture line was clear and sharp within one week but blunt and sclerotic due to bone absorption at 2-3 weeks, and might exist for a long time. All patients had soft tissue swelling and paranasal sinus effusion at 1-2 weeks after injury. Air might gather in the adjacent soft tissues and/or intracranially within 3 days of injury if the fracture involved the frontal or other sinuses. Twelve of the 42 patients (28.6%) had intracranial hematoma, and five (11.9%) had epidural effusion. Subarachnoid hemorrhage was mostly absorbed within one week while epidural hematoma was completely absorbed over 3 weeks. Significant changes (P < 0.05) in the fracture lines, effusion of paranasal sinuses, soft tissue swelling and pneumocephalus were observed during the study period. For patients with medial orbital wall fractures, the fracture line was sharp and clear at early stages with concurrent sphenoid sinus effusion, and the fracture line became depressed 3 weeks later with disappearance of sphenoid sinus effusion. Significant changes (P < 0.05) were observed in the sharp fracture line, soft tissue swelling, sphenoid sinus effusion and smooth depression at fracture sites. For nasal fractures, the fracture line was sharp and clear at early stages with concurrent soft tissue swelling which disappeared one week later. The fracture line became smooth three weeks later. A significant (P < 0.05) difference was demonstrated in the changes of fracture line and soft tissue swelling with time. In conclusion, facial fractures have some dynamic alterations with time and identification of these characteristics may help reaching a correct clinical diagnosis with regard to fracture severity and time.

Potential therapeutic use of relaxin in accelerating closure of cranial bone defects in mice.

Bone fractures are associated with considerable morbidity and increased mortality. A major limitation to healing is lack of bone blood flow, which is impaired by physical disruption of intraskeletal and/or periosteal vasculature by the fracture. Thus, pharmacological interventions are needed to improve osseous blood flow, thereby accelerating bone fracture closure. Relaxin is secreted by the ovary and circulates in rodents and humans during pregnancy. Because relaxin might benefit bone fracture healing by stimulating angiogenesis, vasculogenesis (and potentially osteogenesis) through mobilization and activation of bone marrow progenitor cells, and by increasing blood flow via vasodilation, we investigated whether relaxin administration would accelerate closure of a calvarial defect in mice. Whether administered systemically by osmotic pump or locally by collagen scaffolds for ~2 week period after lesioning, relaxin did not accelerate bone healing. Despite implementing relaxin doses that reached plasma concentrations spanning the physiological to supraphysiological range, testing the closure of two different sizes of calvarial lesions, allowing for different intervals of time from instigation of cranial lesion to euthanasia, and investigating mice of different ages, we did not observe a significant benefit of relaxin in bone lesion healing. Nor did we observe stimulation of blood vessel formation in the bone lesion by the hormone. An incidental finding was that relaxin appeared to enhance trabecular bone growth in an uninjured control bone (femur). Although the results of this study were not supportive of a therapeutic benefit for relaxin on calvarial defect closure, future investigation is needed employing different animal species and experimental models of bone fracture.

Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs. METHODS: The expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining. RESULTS: We observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3' untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis. CONCLUSIONS: Our results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Osteointegration in cranial bone reconstruction: a goal to achieve.

BACKGROUND: The number of cranioplasty procedures is steadily increasing, mainly due to growing indications for decompressive procedures following trauma, tumor or malformations. Although autologous bone is still considered the gold standard for bone replacement in skull, there is an urgent need for synthetic porous implants able to guide bone regeneration and stable reconstruction of the defect. In this respect, hydroxyapatite scaffolds with highly porous architecture are very promising materials, due to the excellent biocompatibility and intrinsic osteogenic and osteoconductive properties that enable deep bone penetration in the scaffold and excellent osteointegration. Osteointegration is here highlighted as a key aspect for the early recovery of bone-like biomechanical performance, for which custom-made porous hydroxyapatite scaffolds play a major role. There are still very few cases documenting the clinical performance of porous scaffolds following cranioplasty. METHODS: This paper reports 2 clinical cases where large cranial defects were repaired by the aid of porous hydroxyapatite scaffolds with customized shapes and 3D profiles (Fin-Ceramica, Faenza, Italy). RESULTS: In the long term (i.e., after 2 years), these scaffolds yielded extensive osteointegration through formation and penetration of new organized bone. CONCLUSIONS: These results confirm that porous hydroxyapatite scaffolds, uniquely possessing chemico-physical and morphological/mechanical properties very close to those of bone, can be considered as a tool to provide effective bone regeneration in large cranial bone defects. Moreover, they may potentially prevent most of the postsurgical drawbacks related to the use of metal or plastic implants.

In vivo gene activity of human mesenchymal stem cells after scaffold-mediated local transplantation.

Functional activation of stem cells after transplantation is a main concern in stem cell therapy. For local transplantation, mesenchymal stem cells (MSCs) are usually administered via scaffolds, either by direct implantation or after preculturing of cells, and it is unclear which is better for the activation of transplanted cells. In this study, we investigated the in vivo gene expression activity of human MSCs (hMSCs) transplanted into calvarial defects either directly post-seeding on collagen sponges (Group 1) or after overnight in vitro culturing post-seeding (Group 2). Real-time reverse transcription-polymerase chain reaction at days 7 and 14 after transplantation identified a time-dependent, rapid decrease in gene expression by the hMSCs, which in Group 1 was slightly more attenuated than in Group 2. Both groups exhibited a limited range of human-specific gene expression, which involved type I collagen (ColI), fibronectin, stromal cell-derived factor (SDF-1), and osteoprotegerin. Among these, ColI expression was the most efficient, with higher levels in Group 1 than Group 2. There was a lack of evidence for the expression of osteoblast differentiation-related markers or trophic factors, while resident cells showed clear expression of those genes. Rat-specific ß-actin expression in Group 2 was least among the scaffold control, Group 1, and Group 2, and this pattern was repeated in the expression of other rat osteogenic genes. Group 1 transplants positively influenced the osteogenic process of the defect tissue in part, and rat IGF-1 expression was significantly increased in Group 1. This tendency of gene expression by hMSCs in a rat model was very similar to what was observed in transplantations using immunodeficient mice. The current study showed that a main gene expressed by transplanted hMSCs during the initial weeks following transplantation is ColI, with a lack of differentiation-related markers or growth factor expression by hMSCs. Our data suggest that direct transplantation of hMSCs loaded on a collagen sponge is more efficient for gene activation in transplanted hMSCs, and more favorable to the local host tissue than transplantation after preculturing of cells.

Influence of BONITmatrix(®) and OSSA NOVA on the expression of bone specific genes.

The aim of this study was to examine the effects of BONITmatrix(®) and OSSA NOVA bone graft materials on osteogenic differentiation. For this, mRNA expression of Alpl, Acp5, Bglap, Phex and Runx2 was analysed using quantitative RT-PCR in surgically created defects to the cranium of adult male rats. Cranial samples were collected after implantation of BONITmatrix(®) or OSSA NOVA scaffolds into these defects for 4 weeks and gene expressions of Alpl, Acp5, Bglap, Phex and Runx2 were measured by quantitative RT-PCR. Real-time PCR analyses showed significantly higher expressions of Acp5, Alpl and Bglap only in the group treated with BONITmatrix(®) compared to untreated controls. No changes in transcript expression of Phex and Runx2 were detectable between the analysed groups. Furthermore, in rats treated with OSSA NOVA the same transcript level of all tested genes was detected as shown in controls. In conclusion, it can be stated that only the granular dosage form of BONITmatrix(®) is able to stimulate osteoblast differentiation, whereas OSSA NOVA has no influence on the gene expression of osteoblasts.

A novel bioactive vitroceramic presents similar biological responses as autogenous bone grafts.

Bioactive glasses represent an interesting class of bone substitute's biomaterials. The present study investigated the repair of bone defects filled with a novel bioactive vitroceramic (Biosilicate(®)), alone or in association with particulate autogenous bone grafts in calvaria defects of rabbits. After 7, 14, and 30 days the specimens were retrieved for histological, histomorphometric and immunohistochemistry analysis. Satisfactory bone formation was observed in all groups, and direct bone-biomaterial surface was noted. Histomorphometric assessment did not show statistically significant differences in bone formation among the groups and periods, except for BG group at day 14. Immunoexpression of Runx-2 was similar among the groups containing the graft and the biomaterial, being more intense than in control group. Similar result was observed for VEGF expression, especially in the last experimental period. These results revealed that Biosilicate(®) presented a favorable behavior, comparable to autogenous bone graft.

Surface immobilization of MEPE peptide onto HA/ß-TCP ceramic particles enhances bone regeneration and remodeling.

Calcium phosphate ceramics have been widely used as scaffolds for bone regeneration. Here, to improve the osteogenic potential of hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) and to apply the bioactive peptide in situ, matrix extracellular phosphoglycoprotein (MEPE) peptide, which has been shown to stimulate osteoblast differentiation, was covalently and directionally immobilized on HA/ß-TCP particles. The free-hydroxyl groups on the surface of the HA/ß-TCP particles were sequentially conjugated with APTES, PEG-(SS)(2), and the synthetic MEPE peptide. Using FTIR and XPS, immobilization of the MEPE peptide on the HA/ß-TCP was confirmed. Implantation of the MEPE peptide-immobilized HA/ß-TCP into calvarial defect and subsequent analyses using a micro CT and histology showed significant bone regeneration and increased bone area (9.89-fold) as compared to that of unmodified HA/ß-TCP. Moreover, tartrate-resistant acid phosphatase-positive osteoclasts were observed in regenerated bone by the MEPE peptide-immobilized HA/ß-TCP, indicating that the bones newly formed by the MEPE peptide-immobilized HA/ß-TCP are actively remodeled by osteoclasts. Therefore, our data demonstrate that MEPE peptide immobilization onto the HA/ß-TCP surface stimulates bone regeneration associated with physiological bone remodeling.

The effect of an octacalcium phosphate co-precipitated gelatin composite on the repair of critical-sized rat calvarial defects.

This study was designed to investigate the extent to which an octacalcium phosphate/gelatin (OCP/Gel) composite can repair rat calvarial critical-sized defects (CSD). OCP crystals were grown with various concentrations of gelatin molecules and the OCP/Gel composites were characterized by chemical analysis, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), selected area electron diffraction (SAED) and mercury intrusion porosimetry. The OCP/Gel composite disks received vacuum dehydrothermal treatment, were implanted in Wistar rat calvarial CSD for 4, 8 and 16 weeks, and then subjected to radiologic, histologic, histomorphometric and histochemical assessment. The attachment of mouse bone marrow stromal ST-2 cells on the disks of the OCP/Gel composites was also examined after 1 day of incubation. OCP/Gel composites containing 24 wt.%, 31 wt.% and 40 wt.% of OCP and with approximate pore sizes of 10-500 µm were obtained. Plate-like crystals were observed closely associated with the Gel matrices. TEM, XRD, FTIR and SAED confirmed that the plate-like crystals were identical to those of the OCP phase, but contained a small amount of sphere-like amorphous material adjacent to the OCP crystals. The OCP (40 wt.%)/Gel composite repaired 71% of the CSD in conjunction with material degradation by osteoclastic cells, which reduced the percentage of the remaining implant to less than 3% within 16 weeks. Of the seeded ST-2 cells, 60-70% were able to migrate and attach to the OCP/Gel composites after 1 day of incubation, regardless of the OCP content. These results indicate that an OCP/Gel composite can repair rat calvarial CSD very efficiently and has favorable biodegradation characteristics. Therefore, it is hypothesized that host osteoblastic cells can easily migrate into an OCP/Gel composite.

Intravital microscopic studies of angiogenesis during bone defect healing in mice calvaria.

PURPOSE: Due to the great availability of specific antibodies, gene-targeted animals and knockout strains, mouse models came into the focus of musculoskeletal research. Herein, we introduce a calvarian defect model in mice that allows the repetitive analysis of blood vessel formation during bone repair by intravital microscopy. METHODS: The right parietal calvaria of 20 adult CD-1 mice were exposed by skin excision. Under continuous irrigation, a circular defect (Ø0.75 mm) was drilled into the calvarium without penetrating the inner cortical shell. A circular glass (Ø12 mm; thickness 0.15 mm) was fixed by two microscrews (M1; length 2mm) to cover the bone defect. Angiogenesis was analysed by intravital microscopy at days 0, 3, 6, 9, 12, 15, 18 and 21. In addition, bone repair was evaluated by histomorphometry at days 3, 6, 9 and 15. Immunohistochemical stainings for the angiogenic growth factor vascular endothelial growth factor (VEGF) and the cell proliferation marker proliferating cell nuclear antigen (PCNA) were performed to assess angiogenic and proliferative activity during healing of the calvarian defect. RESULTS: Histomorphometry showed a typical pattern of intramembranous bone repair. Osseous bridging of the defect was observed at day 9. This was associated with the formation of a neo-periosteum, which covered the new woven bone and contained a dense network of newly formed blood vessels. At day 9, particularly cells of the neo-periosteum showed intense staining for VEGF, whilst PCNA-positive staining was found mainly in osteoblasts. At day 15, the major fraction of fibrous tissue was replaced by bone undergoing extensive remodelling. Intravital microscopy revealed an increase of vascular density between days 3 and 15. Blood vessel diameters showed an increase between days 3 and 9 and a subsequent decrease between days 9 and 21. CONCLUSIONS: The present calvarian defect model provides a powerful tool to evaluate the process of angiogenesis during intramembranous bone repair in mice.

Icariin: a potential osteoinductive compound for bone tissue engineering.

To effectively treat bone diseases using bone regenerative medicine, there is an urgent need to develop safe and cheap drugs that can potently induce bone formation. Here, we demonstrate the osteogenic effects of icariin, the main active compound of Epimedium pubescens. Icariin induced osteogenic differentiation of preosteoblastic cells. The combination of icariin and a helioxanthin-derived small compound synergistically induced osteogenic differentiation of MC3T3-E1 cells to a similar extent to bone morphogenetic protein-2. Icariin enhanced the osteogenic induction activity of bone morphogenetic protein-2 in a fibroblastic cell line. Mineralization was enhanced by treatment with a combination of icariin and calcium-enriched medium. The in vivo anabolic effect of icariin was confirmed in a mouse calvarial defect model. Eight-week-old male C57BL/6N mice were transplanted with icariin-calcium phosphate cement (CPC) tablets or CPC tablets only (n = 5 for each), and bone regeneration was evaluated after 4 and 6 weeks. Significant new bone formation was observed in the icariin-CPC group at 4 weeks, and the new bone thickness had increased by 6 weeks. Obvious blood vessel formation was observed in the icariin-induced new bone. Treatment of senescence-accelerated mouse prone 1 and senescence-accelerated mouse prone 6 models further demonstrated that icariin was able to enhance bone formation in vivo. Therefore, icariin is a strong candidate for an osteogenic compound for use in bone tissue engineering.

Mechanical stress-related calvaria bone augmentation by onlayed octacalcium phosphate-collagen implant.

Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-kappaB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided.

Linking of bone morphogenetic protein-2 to resorbable fracture plates for enhancing bone healing.

OBJECTIVE: To test whether bone morphogenetic protein (BMP)-2 may be covalently linked to resorbable fracture repair plates using an ester-hydrolysis reaction and determining whether the linked compound can facilitate bone growth. STUDY DESIGN: Laboratory in vitro experiments. METHOD: Resorbable fracture repair plates were partially hydrolyzed using varying concentrations of acid or base. This intermediate was then reacted with EDAC (1-ethyl-3[-3-dimethylamino propyl carbodiimide) to form an EDAC intermediate, which was then reacted with either horseradish peroxidase (HRP), interleukin (IL)-2, or BMP-2. Compound binding to the plate was confirmed by immunofluorescent staining. Confirmation of protein function was determined by the following assays: HRP's ability to cleave peroxide, IL-2's ability to stimulate lymphocytes, and BMP-2's ability to stimulate C3H10T1/2 cells to generate alkaline phosphatase. RESULTS: Three compounds (HRP, IL-2, and BMP-2) were successfully linked to plates as confirmed by immunofluorescence staining or functional testing. Compounds demonstrated better covalent linking to plates under basic conditions. HRP, IL-2, and BMP-2 retained function after binding as measured by cleaved peroxide levels, lymphocytes proliferation, and alkaline phosphatase production. CONCLUSIONS: Covalent linking of compounds such as HRP, IL-2, and BMP-2 to resorbable plates is possible and represents a novel protein delivery technique. BMP-2 covalently linked to resorbable plates may be used to facilitate bone healing. Covalent linking of compounds to plates represents a novel method for delivering concentrated levels of growth factors to a specific site and potentially extending their half-life. Further investigation into this application for bone healing may lead to quicker healing.

Expression of bone matrix proteins during de novo bone formation using a bovine collagen and platelet-rich plasma (prp)--an immunohistochemical analysis.

This animal study (domestic pig) examined the bone formation after filling defined defects with autogenous bone or a collagen lyophilisat in combination with Platelet-rich-plasma (PRP) by evaluating bone matrix proteins. Six groups, both materials with and without PRP in two concentrations (+ 1, + 2) were compared to untreated bone by means of immunohistochemistry at 2, 4, 12 and 26 weeks. BMP-2 expression was increased at 2 weeks in the collagen + 1 group and after 4 weeks in the collagen + 1 and + 2 group. Collagen-I expression was increased at 2 weeks in all collagen groups. After 4 weeks raised levels were observed after adding the higher concentrated PRP to bone and the collagen material. Osteocalcin expression was enhanced at 2 weeks in all collagen groups and the autogenous bone + PRP1 group, after 4 weeks in the bone and collagen + 2 groups. At 12 weeks higher values were observed after adding higher concentrated PRP to bone. Osteonectin and especially osteopontin were confirmed to be effective markers of early bone formation in all specimens. The described setting allows to combine established techniques (microradiography, light microscopy) with approaches to explore the underlying biology (immunohistochemistry) on the same specimen.

Circulating levels of interleukin-6 and its soluble receptor in patients with head injury and fracture.

There is evidence that fractures heal more rapidly in patients with head injury. We measured the circulating level of interleukin-6 (IL-6) and its soluble receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) in serum from patients who had sustained a head injury with and without fracture and compared these with levels found in control subjects. Within 12 hours of injury the serum level of IL-6 was significantly higher in patients with head injury and fracture compared with the control group. Levels of IL-6 were also significantly higher in patients with head injury and fracture compared with fracture only. While there was no significant difference in circulating levels of sIL-6R in the initial samples they were increased one week after surgery in patients with head injury and fracture and with head injury only. In addition, reduced levels of sgp130 in patients with head injury with and without fracture indicated a possible reduction of the inhibitory effect of this protein on the activity of IL-6. Our study suggests that IL-6 may be involved in altered healing of a fracture after head injury.

Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy.

The clinical significance of hydroxyapatite (HAP) as a bone substitute has become apparent in recent years and bone morphogenetic protein (BMP) a substance which induces bone has attracted much attention. In this study, a 1.2 cm diameter bone defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAP after completion of hemostasis and the resultant bone formation was analyzed histopathologically. The amounts of bone formation was compared BMP-2 cDNA plasmids were not combined with cationic liposomes as a vector. Four groups of rabbits were compared. In the HAP group the cranial bone defect was treated with HAP containing 40 microg of liposomes and a dummy gene (PU). The BMP gene HAP group was treated with HAP soaked in liposomes and 10 microg of the BMP-2 gene. In addition, a group was treated with the gene without implanting HAP. Bone formation on the cranial defects was evaluated 3, 6 and 9 weeks after the operation, by X-ray and histopathological examinations. Three weeks after the operation there was vigorous bone formation in the cranial defect in the group which received the BMP-2 gene without HAP, and complete ossification was observed at 9 weeks. In the group which received HAP containing the BMP-2 gene, although new bone formation was evident surrounding the scaffold 3 weeks post-operation, the induced bone tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. These results confirm the clinical usefulness of gene therapy for bone formation, using the BMP-2 gene combined with cationic liposomes as a vector. It is possible that the effects of administering the BMP-2 gene will be improved by specializing the microstructure of scaffold for gene therapy.

Expression of TGF-beta in region of bone defect repaired by collagen/nano-beta-tricalcium phosphate composite artificial bone.

The distribution and function of transforming growth factor-beta (TGF-beta) in the region of bone defect repaired by collagen/nano-beta-tricalcium phosphate composite artificial bone (Co/N-TCP) and the ability of Co/N-TCP recruiting osteoblasts to precipitate the repair of bone defect were investigated. Twenty-four domestic rabbits were operated on bilateral cranial bone to create an experimental bone defect of 8.0 mm in diameter through the whole bone. On the left, Co/N-TCP was implanted as experimental group, but on the right, Co/TCP was implanted as control group. At 2nd, 4th, 8th, 12th week after operation, all animals were sacrificed and the implanted materials with surrounding bone were taken out. Immunohistochemical staining was performed for TGF-beta assay by avidin-biotin complex method (SABC). Simultaneously, TGF-beta was quantitatively analyzed by HPIAS-1000 imaging analysis system. The immunohistochemical staining for TGF-beta revealed that osteoblasts and immature osteocytes highly expressed TGF-beta. Diffused TGF-beta positive staining particles appeared in the mesenchymal and fibrous-tissue. There was no significant difference in the TGF-beta positive staining between two groups in the medial region to original osseous beds at different time points (P > 0.05). However, in distal original osseous bed of the defected region, the positive expression of TGF-beta in the Co/N-TCP group was significantly stronger than in the control group (P < 0.05 or 0.01). The Co/N-TCP has good bioactivities and ability of stimulating and conducting TGF-beta to aggregate and precipitate the healing of bone defect.

Expression of bone morphogenetic proteins during membranous bone healing.

For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques. In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair.