Hydrolyzed Collagen Tripeptide Promotes Longitudinal Bone Growth in Childhood Rats via Increases in Insulin-Like Growth Factor-1 and Bone Morphogenetic Proteins.

Previous studies have reported that collagen tripeptide (CTP) derived from collagen hydrolysate has various beneficial effects on health by protecting against skin aging and improving bone formation and cartilage regeneration. Collagen-Tripep20TM (CTP20), which is a low-molecular-weight CTP derived from fish skin, contains a bioactive CTP, Gly-Pro-Hyp >3.2% with a tripeptide content >20%. Herein, we investigated the osteogenic effects and mechanisms of CTP20 (<500 Da) on MG-63 osteoblast-like cells and SW1353 chondrocytes. And we measured promoting ratio of the longitudinal bone growth in childhood rats. First, CTP20 at 100 µg/mL elevated the proliferation (15.0% and 28.2%), alkaline phosphatase activity (29.3% and 32.0%), collagen synthesis (1.25- and 1.14-fold), and calcium deposition (1.18- and 1.15-fold) in MG-63 cells and SW1353, respectively. In addition, we found that CTP20 could promote the longitudinal growth and height of the growth plate of the tibia in childhood rats. CTP20 enhanced the protein expression of insulin-like growth factor-1 (IGF-1) in MG-63 and SW1353 cells, and in the growth plate of childhood rats, along with Janus Kinase 2, and signal transducer and activator of transcription 5 activation in MG-63 and SW1353 cells. CTP20 also elevated the expression levels of bone morphogenetic proteins (BMPs) in MG-63 and SW1353 cells and in the growth plates of childhood rats. These results indicate that CTP20 may promote the endochondral ossification and longitudinal bone growth, through enhancing of IGF-1 and BMPs. (Clinical Trial Registration number: smecae 19-09-01).

Enhancement of the effects of intermittent parathyroid hormone (1-34) by bone morphogenetic protein in a rat femoral open fracture model.

BACKGROUND: Nonunion in cases of open fracture is common. Both bone morphogenetic protein 2 (BMP-2) and parathyroid hormone (PTH) have been used to enhance bone healing. We investigated the combination of BMP-2 and PTH and examined the effects on a rat model of open femoral fractures. METHODS: Group I (n = 11) was implanted with control carrier. Group II (n = 12) was implanted with carrier containing 1 µg of recombinant human BMP-2 (rhBMP-2). Group III (n = 12) was implanted with carrier alone, followed by injections of PTH 1-34. Group IV (n = 11) was implanted with carrier containing 1 µg of rhBMP-2, followed by injections of PTH 1-34. Group V (n = 11) was implanted with carrier containing 10 µg of rhBMP-2. Group VI (n = 11) was implanted with carrier containing 10 µg of rhBMP-2, followed by injections of PTH 1-34. Rats were euthanized after 8 weeks, and their fractured femurs were explanted and assessed by manual palpation, radiographs, micro-computerized tomography, and histological analysis. RESULTS: Manual palpation tests showed that the fusion rates of groups III (66.7%), IV (63.6%), V (81.8%), and VI (81.8%) were considerably higher than those of group I. Groups V and VI had higher radiographic scores compared to group I. Micro-CT analysis revealed enhanced bone marrow density expressed as bone volume/tissue volume in groups V (61.88 ± 3.16%) and VI (71.14 ± 3.89%) versus group I (58.26 ± 1.86%). A histological analysis indicated that group VI had enhanced remodeling. CONCLUSION: The combination of abundant rhBMP-2 and PTH enhanced bone healing and remodeling of newly formed bone in a rat femoral open fracture model.

Photobiomodulation Therapy in Bone Repair Associated with Bone Morphogenetic Proteins and Guided Bone Regeneration: A Histomorphometric Study.

OBJECTIVE: To evaluate the efficacy of photobiomodulation for bone repair of critical surgical wounds with implants of bone morphogenetic proteins (BMPs) and bovine biological membranes, using histological and histomorphometric analysis. BACKGROUND: Tissue engineering has been developing rapidly through the use of various biomaterials for the treatment of bone defects, such as mechanical barriers consisting of biological membranes and implants of biomaterials for bone supply. MATERIALS AND METHODS: Thirty-two male rats were divided into four groups (n = 8): group I-C: control; group II-PT: photobiomodulation therapy; group III-PM: Gen-Pro® BMPs+Gen-Derm® membrane; and group IV-PMPT: Gen-Pro® BMPs+Gen-Derm® membrane+photobiomodulation therapy. A 3 mm bone cavity was performed in the upper third of the lateral surface of the right rat femur to obtain a bone defect considered to be critical. The irradiated groups received seven applications of AlGaAs diode laser 830 nm, P = 40 mW, continuous wave (CW) emission mode, f ∼ 0.6 mm, 4 J/cm2 per point (north, south, east, and west) at 48 h intervals, for a total of 16 J/cm2 per session (final dose: 112 J/cm2). Bone repair was evaluated at sacrifice 15 and 30 days after the procedure. The specimens were embedded in paraffin and stained with hematoxylin and eosin and Picrosirius for analysis by light microscopy and by the Leica interactive measurement module software. Statistical analysis was performed (p < 0.05%). RESULTS: Histological analysis confirmed the histomorphometric results, with the experimental groups showing bone neoformation of significantly higher quality and quantity at the end of 30 days compared with the control group. CONCLUSIONS: Photobiomodulation therapy was effective for bone repair mainly when associated with BMPs and a biological membrane. The results of this study are promising and stimulate further scientific and clinical research.

Biologically synthesized titanium oxide nanostructures combined with morphogenetic protein as wound healing agent in the femoral fracture after surgery.

The aim of the present study is to develop novel approach for the green synthesis of titanium oxide nanoparticles (TiO2 NPs) using Eichhornia crassipes extract and calcined at different temperatures for evaluate the wound healing activity in the femoral fracture. The synthesized TiO2 are formed different (plate and rod-like) nanostructures at various calcination temperatures. These samples were characterized by X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FTIR), Field emission scanning electron microscope (FE-SEM) and transmission electron microscope (TEM). Microscopic studies of TiO2 NPs revealed that the synthesized TiO2 NPs are formed well-defined rod-like structures at 400 °C with size ranged from 200 nm to 500 nm. The characterized plate and rod-like TiO2 NPs are combined with human morphogenetic protein (HbMP) to improving its wound healing activity and osteoblast properties on femoral fractures. The biocompatibility was tested by using human bone marrow mesenchymal stem cells (BMSC) cells and antibacterial efficacy analyzed using human pathogenica bacteria Staphylococcus aureus and Escherichia coli through agar well diffusion assay. The green synthesized rod-like TiO2 NPs combined with HbMP has been exhibited effective bone fusion behaviors with biomechanical properties and also improved antibacterial activity against pathogenic bacteria. From this study results, it is suggested that green synthesized TiO2NPs could be used effectively in biomedical application.

Biologic Approaches to Problems of the Hand and Wrist.

Orthobiologics are not used as frequently in the hand and wrist as in other sites. The most frequently reported is the use of bone morphogenetic protein for the treatment of Kienböck disease. Animal studies have described improved tendon healing with the use of platelet-rich plasma (PRP), but no clinical studies have confirmed these results. PRP has been reported to produce improvements in the outcomes of distal radial fractures and osteoarthritis of the trapeziometacarpal in small numbers of patients. The use of orthobiologics in the hand and wrist are promising, but clinical trials are necessary to establish efficacy and safety.

Suppression of osteoblast-related genes during osteogenic differentiation of adipose tissue derived stromal cells.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.

Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

This "Controversies in Cardiovascular Research" article evaluates the evidence for and against the hypothesis that the circulating blood level of growth differentiation factor 11 (GDF11) decreases in old age and that restoring normal GDF11 levels in old animals rejuvenates their skeletal muscle and reverses pathological cardiac hypertrophy and cardiac dysfunction. Studies supporting the original GDF11 hypothesis in skeletal and cardiac muscle have not been validated by several independent groups. These new studies have either found no effects of restoring normal GDF11 levels on cardiac structure and function or have shown that increasing GDF11 or its closely related family member growth differentiation factor 8 actually impairs skeletal muscle repair in old animals. One possible explanation for what seems to be mutually exclusive findings is that the original reagent used to measure GDF11 levels also detected many other molecules so that age-dependent changes in GDF11 are still not well known. The more important issue is whether increasing blood [GDF11] repairs old skeletal muscle and reverses age-related cardiac pathologies. There are substantial new and existing data showing that GDF8/11 can exacerbate rather than rejuvenate skeletal muscle injury in old animals. There is also new evidence disputing the idea that there is pathological hypertrophy in old C57bl6 mice and that GDF11 therapy can reverse cardiac pathologies. Finally, high [GDF11] causes reductions in body and heart weight in both young and old animals, suggestive of a cachexia effect. Our conclusion is that elevating blood levels of GDF11 in the aged might cause more harm than good.

Complete subchondral bone defect regeneration with a tricalcium phosphate collagen implant and osteoinductive growth factors: a randomized controlled study in Göttingen minipigs.

The restoration and reconstruction of osseous defects close to the joint, constitutes a challenging field for reconstructive surgery. A dual-layer implant of ß-tricalcium phosphate (TCP) and a collagens I/III scaffold was evaluated in a prospective, randomized comparison in a larger animal model. For this purpose, a standardized osteochondral defect was created in the medial facet of the patellar groove in both stifle joints of Göttingen minipigs. Critical-size osseous defects were either left empty (spontaneous healing; group 1; n = 12) or treated with the two-layer TCP collagen implant (group 2; n = 12). In group 3 (n = 12), additional growth factor mixture (GFM) was supplemented (bone morphogenetic proteins 2, 3, 4, 6, 7, and TGF-ß1, 2, 3). Osseous defect regeneration was assessed at 6, 12, and 52 weeks postoperatively (n = 4). Qualitative and quantitative histomorphometric assessment of defect regeneration and bone substitute resorption was conducted by means of light microscopy, fluorescence microscopy, and microradiography. Critical-size defects did not heal spontaneously throughout follow-up (group 1: max. 21.84 ± 2.81% defect area at 52 weeks). The TCP layer of the implant significantly increased the amount of new bone formation with 29.8 ± 9.68% at 6 weeks and 40.09 ± 4.76% at 12 weeks when compared with controls. After 52 weeks, the TCP was almost fully degraded (4.35 ± 3.70%) and the defect was restored with lamellar trabecular bone (31.28 ± 5.02%). Growth factor supplementation resulted in earlier resorption of the TCP implant and faster defect regeneration. The dual-layer TCP collagen implant is suitable to restore subchondral osseous defects. Additional use of GFM increased the resorption of the TCP layer, but did not foster new bone formation.

Effect of BMP-12, TGF-ß1 and autologous conditioned serum on growth factor expression in Achilles tendon healing.

PURPOSE: Achilles tendon ruptures are devastating and recover slowly and incompletely. There is a great demand for biomolecular therapies to improve recovery, yet little is understood about growth factors in a healing tendon. Here, the role of growth factors during tendon healing in a rat model and their reaction to single and multiple growth factor treatment are explored. METHODS: Rat tendons were transected surgically and resutured. The expression of bFGF, BMP-12, VEGF and TGF-ß1 was assessed by immunohistochemical analysis one to 8 weeks after surgery. Paracrine effects of TGF-ß1 or BMP-12 added by adenoviral transfer, as well as the effect of autologous conditioned serum (ACS) on growth factor expression, were evaluated. RESULTS: bFGF, BMP-12 and VEGF expression was highest 1 week after transection. bFGF and BMP-12 declined during the remaining period whereas VEGF expression persisted. TGF-ß1 expression dramatically increased after 8 weeks. ACS treatment increased bFGF (P = 0.007) and BMP-12 (P = 0.004) expression significantly after 8 weeks. Also overall expression of bFGF, BMP-12 and TGF-ß1 regardless of time point was significantly greater than controls with ACS treatment (P < 0.05). Both BMP-12 and TGF-ß1 treatments had no significant effect. No effect was observed in VEGF with any treatment. CONCLUSION: bFGF, BMP-12, VEGF and TGF-ß1 are differentially expressed during tendon healing. Additional BMP-12 or TGF-ß1 has no significant influence, whereas ACS generally increases expression of all factors except VEGF. Staged application of multiple growth factors may be the most promising biomolecular treatment.

Light microscopic description of the effects of laser phototherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration in a rodent model.

We carried out a histological analysis on bone defects grafted with mineral trioxide aggregate (MTA) treated or not with laser, bone morphogenetic protein (BMP), and guided bone regeneration (GBR). Benefits of the use of MTA, laser, BMPs, and GBR on bone repair are well known, but there is no report on their association with laser light. Ninety rats were divided into 10 groups each subdivided into 3. Defects on G II and I were filled with the blood clot. G II was further irradiated with LED. G III and IV were filled with MTA; G IV was further irradiated with laser. G V and VI, the defects filled with MTA and covered with a membrane (GBR). G VI was further irradiated with laser. G VII and VIII, BMPs were added to the MTA and group VIII further irradiated with laser. G IX and X, the MTA + BMP graft was covered with a membrane (GBR). G X was further irradiated with laser. Laser light (λ = 850 nm, 150 mW, 4 J/cm(2) ) was applied over the defect at 48-h intervals and repeated for 15 days. Specimens were processed, cut and stained with H&E and Sirius red and underwent histological analysis. Subjects on group X were irradiated. The results showed different tissue response on all groups during the experimental time. Major changes were seen on irradiated subjects and included marked deposition of new bone in advanced maturation. It is concluded that near infrared laser phototherapy improved the results of the use of the MTA on bone defects.

Treatment with rhBMP12 or rhBMP13 increase the rate and the quality of rat Achilles tendon repair.

Tendon injuries that result in partial or complete tears often come from chronic, repetitive use, or from sudden trauma. In some cases, torn tendons can be repaired, but such repairs often fail to completely restore tendon function. We used global gene expression profiling and histological examination to study tendon repair to elucidate key molecular processes that regulate the rate and quality of tissue restoration. Using a rat Achilles tendon transection model, tissue was collected at 3, 7, 10, and 15 days postinjury. The pattern of gene expression in the repairing tissue paralleled the healing phases of inflammation, matrix formation, and matrix reorganization. Newly formed repaired tissue is characterized by cells expressing many genes associated with tendon formation, thereby potentially distinguishing this repair tissue from other types of repair or scar tissue. Addition of recombinant human bone morphogenic protein (rhBMP)12 or rhBMP13, also known as growth and differentiation factors (GDFs) 6 and 7, 1 day after injury yielded increases in tissue volume, rate of cellular infiltration, and in changes in levels of key mRNAs involved in tendon repair. Altogether, our results indicate that rhBMP12 or rhBMP13 enhance the rate of tendon repair. A better understanding of the key molecular regulators of tendon repair could lead to the development of new therapies for tendon injuries and the identification of diagnostic markers that indicate the status of tendon repair after injury.

Effects of bone morphogenetic proteins on osteoblast cells: vascular endothelial growth factor, calcium, inorganic phosphate, and nitric oxide levels.

PURPOSE: Bone morphogenetic proteins (BMPs) play an important role in the initiation of bone formation by affecting cell growth and differentiation in a variety of cell types including osteoblasts. Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and vasculogenesis, and also, VEGF signaling is important for skeletal development. Nitric oxide (NO), calcium (Ca), and inorganic Phosphate (Pi) are important molecules for cell functions. In this study, the effects of BMP on VEGF, Ca, NO, and Pi levels were investigated in an osteoblast cell culture. MATERIALS: Fifty thousand cells per milliliter were seeded and cultured on graft materials for 24 and 48 hours. Different concentrations of BMPs (combination of BMPs numbered from 1 to 14) were supplemented to the medium. RESULTS: BMP was found to increase VEGF (P = 0.00), Ca (P = 0.02), and Pi (P = 0.00) especially in the first 24 hours. The increase in the NO in the experimental groups were found to be statistically insignificant (P = 0.12). CONCLUSION: Our data state that further investigation should be performed on the effects of BMPs on osteoblast cell membranes and membrane receptors and cell signaling, together with their known effects on early phases of bone and vascular epithelial tissue formation.

Effects of laser photherapy on bone defects grafted with mineral trioxide aggregate, bone morphogenetic proteins, and guided bone regeneration: a Raman spectroscopic study.

We have used Raman analysis to assess bone healing on different models. Benefits on the isolated or combined use of mineral trioxide aggregate, bone morphogenetic proteins, guided bone regeneration and laser on bone repair have been reported, but not their combination. We studied peaks of hydroxyapatite and CH groups on defects grafted with MTA, treated or not with laser, BMPs, and GBR. Ninety rats were divided in 10 groups each, subdivided into three subgroups. Laser (λ850 nm) was applied at every other day for 2 weeks. Raman readings were taken at the surface of the defect. Statistical analysis (CHA) showed significant differences between all groups (p = 0.001) and between Group II and all other (p < 0.001), but not with Group X (p = 0.09). At day 21 differences were seen between all groups (p = 0.031) and between Groups VIII and X when compared with Groups VI (p = 0.03), V (p < 0.001), IV (p < 0.001), and IX (p = 0.04). At the end of the experimental period no significant differences were seen. On regards CH, significant differences were seen at the 15(th) day (p = 0.002) and between Group II and all other groups (p < 0.0001) but not with control. Advanced maturation on irradiated bone is because of increased secretion of calcium hydroxyapatite (CHA) that is indicative of greater calcification and resistance of the bone. We conclude that the association of the MTA with laser phototherapy (LPT) and/or not with GBR resulted in a better bone repair. The use of the MTA associated to IR LPT resulted in a more advanced and quality bone repair.

Morphometric and histological analysis of low-power laser influence on bone morphogenetic protein in bone defects repair.

Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-beta (TGF-beta) superfamily. The objective of this study was to determine how gallium-aluminum-arsenium (GaAlAs) 650 nm laser influenced the action of BMPs on bone defects created in rat femurs. The sample consisted of 24 male albino Wistar rats. Group 1 was composed of rats with bone defects filled with bone-inducing substance, with the application of low-power laser. Group 2 contained rats with bone defects filled with a bone-inducing substance, without the application of low-power laser. Group 3 rats had bone defects not filled with a bone-inducing substance, with the application of low-power laser. Group 4 rats had bone defects and no treatment (control group). A bone defect was produced with drills. In groups 1 and 2 the defects were filled with a bone-inducing substance. The animals were treated with GaAlAs (50 mW) laser, energy density 4 J/cm(2), for 80 ss on a 1 cm(2) area. Groups 2 and 4 were used as control. Bone samples were removed for histological procedures and morphometric analysis on the 7th, 14th and 21st days after surgery. Results obtained were subjected to statistical analysis. Rejection level for the null hypothesis was 0.05. Statistical differences were found in the comparison between group 1 (G1), G2, G3 and G4 [analysis of variance (ANOVA); P < 0.0134]. There was a statistically significant correlation between groups 1 and 4 (P < 0.01). The results of other correlations by Tukey's post-hoc test were: group 1 vs group 3 (P = 0.341), group 1 vs group 2 (P = 0.862), group 2 vs group 4 (P = 0.061), group 2 vs group 3 (P = 0.744), and group 3 vs group 4 (P = 0.249). We concluded that the association of low-power laser with a bone-inducing substance produced better results than when low-power laser or BMPs were used alone.

Development of high-throughput screening system for osteogenic drugs using a cell-based sensor.

To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.

Does the use of laser photobiomodulation, bone morphogenetic proteins, and guided bone regeneration improve the outcome of autologous bone grafts? An in vivo study in a rodent model.

OBJECTIVE: The aim of the present investigation was to histologically assess the effect of laser photobiomodulation (LBPM) on the repair of autologous bone grafts in a rodent model. BACKGROUND DATA: A major problem in modern dentistry is the recovery of bone defects caused by trauma, surgical procedures, or pathologies. Several types of biomaterials have been used to improve the repair of these defects. These materials are often associated with procedures of guided bone regeneration (GBR). MATERIALS AND METHODS: Twenty four animals were divided into four groups: group I (control); group II (LPBM of the bone graft); group III (bone morphogenetic proteins [BMPs] + bone graft); and group IV (LPBM of the bed and the bone graft + BMPs). When appropriate the bed was filled with lyophilized bovine bone and BMPs used with or without GBR. The animals in the irradiated groups received 10 J/cm(2) per session divided over four points around the defect (4 J/cm(2)), with the first irradiation immediately after surgery, and then repeated seven times every other day. The animals were humanely killed after 40 d. RESULTS: The results showed that in all treatment groups, new bone formation was greater and qualitatively better than the untreated subjects. Control specimens showed a less advanced repair after 40 d, and this was characterized by the presence of medullary tissue, a small amount of bone trabeculi, and some cortical repair. CONCLUSION: We conclude that LPBM has a positive biomodulatory effect on the healing of bone defects, and that this effect was more evident when LPBM was performed on the surgical bed intraoperatively, prior to the placement of the autologous bone graft.

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells.

BACKGROUND: Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. METHODS: Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and beta-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. RESULTS: ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. CONCLUSION: Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.

Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants.

The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with (125)I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.

BMP-2 and bFGF in an irradiated bone model.

INTRODUCTION: Basic fibroblast growth factor (bFGF) is considered to enhance angiogenesis and to support bone formation in the presence of vital bone cells. Bone morphogenetic protein-2 (rhBMP-2) is known to induce bone formation. The aim of this study was to analyze the effect of bFGF and rhBMP-2 in the irradiated mandible. MATERIAL AND METHODS: The right mandibles of 24 rats were irradiated with a single dose of 20 Gy at a high-dose-rate (HDR) after loading machine (bio effective equivalent dose to ca. 45 x 2 Gy). After 12 weeks 100 microg rhBMP-2 (n=6 animals, group 1), 100 microg bFGF (n=6 animals, group 2) and 100 microg rhBMP-2 plus 100 microg bFGF (n=6 animals, group 3) were injected along the right mandible (left mandible: no irradiation, no growth factor). Another 6 animals (group 4) remained untreated after the irradiation. After another 7 weeks the specimens were examined by non-decalcified histology. RESULTS: Bone apposition of the experimental versus control sides was not statistically significantly different when one of the growth factors was applied alone (rhBMP-2: p=0.917; bFGF: p=0.345). Average bone apposition was significantly decreased on the experimental sides of group 3 (rhBMP-2+bFGF: p=0.046) and group 4 (p=0.008). Average bone densities were unaffected in all settings (for all p>0.1). CONCLUSIONS: The application of bFGF and the application of rhBMP-2 alone did result in predictable bone generation in the irradiated mandible with the bone apposition being equal to that of the non-irradiated side. The application of both growth factors together or none at all after irradiation results in significantly reduced bone apposition.

The effect of combined application of TGFbeta-1, BMP-2, and COLLOSS E on the development of bone marrow derived osteoblast-like cells in vitro.

This study investigated the combined application of Transforming Growth Factor beta-1 (TGFbeta-1) and Bone Morphogenetic Protein-2 (BMP-2) to stimulate osteogenic expression in vitro. TGFbeta-1 and BMP-2 fulfill specific roles in the formation of new bone. COLLOSS E, a bone-derived collagen product containing a variety of naturally occurring growth factors, was also used. Growth factors were administered to osteoblast-like cells from rat bone marrow (RBM). Proliferation and differentiation were monitored up to 24 days, by measuring total DNA content, alkaline phosphatase activity, and calcium content. Genetic expression of a set of differentiation markers at day 7 was measured by Q-PCR. Adding BMP-2 alone induced high proliferation rates, compared to the growth factor supplemented groups, and it induced high differentiation rates, compared to the control group. Adding TGFbeta-1 combined with BMP-2, TGFbeta-1 alone, or COLLOSS E resulted in a significant decrease in proliferation rate, but an increase in differentiation rate, compared to the control group. Additive or synergistic effects of application of TGFbeta-1 and BMP-2 were not observed. The observed effects of COLLOSS E mainly resembled those of TGFbeta-1 application alone. It can be concluded that BMP-2 is the most suitable candidate for osteogenic stimulation of RBM cells in these settings.